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EC number: - | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
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- Endpoint summary
- Stability
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-04-04 to 2019-06-19
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use, June 2012
- Version / remarks:
- 2012
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Reaction mass of sodium bis(2-ethylhexyl) phosphate and sodium bis(2-ethylhexyl) phosphate
- Molecular formula:
- C8H19O4P.2Na - C16H35O4P.Na
- IUPAC Name:
- Reaction mass of sodium bis(2-ethylhexyl) phosphate and sodium bis(2-ethylhexyl) phosphate
Constituent 1
Method
- Target gene:
- The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).
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:
- induced rat liver S9
- Test concentrations with justification for top dose:
- At the preparation of the test item stock solution a correction (multiplier) factor of 1.953 (1/0.512=1.953) based on the purity of 51.2% was taken into consideration.
Based on the cytotoxicity and solubility results of the preliminary concentration range finding test (informatory toxicity test) and based on the recommendations in OECD 471 guideline, the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests:
3200; 1600; 800; 320; 128; 51.2 and 20.5 μg/plate. - Vehicle / solvent:
- - Vehicle/solvent used: ultrapure water
- Justification for choice of solvent/vehicle: suitable vehicle according to the guideline and sufficient solubility of the test item.
- Justification for percentage of solvent in the final culture medium: as recommended in the guideline
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, NPD (TA 98; without S9; 4 µg/plate); 2-aminoanthracene, 2AA (all strains, with S9; 2 µg/plate (S. typhimurium), 50 µg/plate (E.coli)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
DURATION
- Preincubation period: 20 min at 37 °C (bacterial culture and the S9 Mix or phosphate buffer)
- Exposure duration: 48 hours in the dark at 37 °C
NUMBER OF REPLICATIONS:
3
DETERMINATION OF CYTOTOXICITY
A dose level is considered toxic if:
- the reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs
- other: The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate. - Rationale for test conditions:
- According to guidelines.
- Evaluation criteria:
- The colony numbers on the control, positive control and the test plates were determined, the mean values, standard deviations and the mutation rates were calculated.
Mutation Rate = Mean revertants at the test item (or control) treatments / Mean revertrants of vehicle control
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 TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain 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 was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.
Criteria for a Negative Response:
A test article is considered non-mutagenic in this bacterial reverse mutation assay 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.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- 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:
- 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 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- 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:
- 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:
- 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: yes
- Data on osmolality: yes
- Possibility of evaporation from medium: no
- Water solubility: sufficient soluble
- Precipitation and time of the determination: no
RANGE-FINDING/SCREENING STUDIES: yes
STUDY RESULTS
- Concurrent vehicle negative and positive control data
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value
Ames test:
- Signs of toxicity : please refer to "any other information on results including tables"
- Individual plate counts: please refer to "any other information on results including tables"
- Mean number of revertant colonies per plate and standard deviation : please refer to "any other information on results including tables"
HISTORICAL CONTROL DATA
- Positive historical control data: please refer to "any other information on results including tables"
- Negative (solvent/vehicle) historical control data: please refer to "any other information on results including tables"
Any other information on results incl. tables
Table 1: Summary of Signs of Cytotoxicity in the Plate Incorporation and Pre-Incubation Tests
Plate Incorporation Test |
||||||||||
Concentrations |
Salmonella typhimurium |
Escherichia coliWP2uvrA |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
|||||||
‑S9 |
+S9 |
‑S9 |
+S9 |
‑S9 |
+S9 |
‑S9 |
+S9 |
‑S9 |
+S9 |
|
3200 |
B<< |
B<< |
B<< |
B<< |
B<< |
SB< |
B0 |
B0 |
B<< |
SB<< |
1600 |
* |
– |
SB<< |
<< |
– |
– |
SB< |
* |
* |
<< |
800 |
– |
* |
<< |
– |
– |
– |
* |
– |
* |
– |
320 |
– |
* |
– |
– |
– |
– |
– |
– |
* |
– |
128 |
– |
– |
* |
<<* |
– |
– |
– |
– |
– |
– |
51.2 |
– |
– |
– |
– |
– |
– |
– |
– |
* |
– |
20.5 |
– |
* |
– |
– |
– |
– |
– |
– |
* |
– |
Pre-Incubation Test |
||||||||||
Concentrations |
Salmonella typhimurium |
Escherichia coliWP2uvrA |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
|||||||
‑S9 |
+S9 |
‑S9 |
+S9 |
‑S9 |
+S9 |
‑S9 |
+S9 |
‑S9 |
+S9 |
|
3200 |
A |
A |
A |
A |
A |
A |
A |
A |
B<< |
SB, PP<< |
1600 |
B0 |
B0 |
B0 |
B0 |
B0 |
B0 |
B0 |
B<< |
SB<< |
< |
800 |
B<< |
SB<< |
B0 |
B<< |
SB< |
SB< |
B< |
SB0 |
* |
– |
320 |
SB<< |
– |
SB# |
– |
SB< |
– |
B< |
SB |
– |
– |
128 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
51.2 |
– |
– |
– |
– |
– |
* |
– |
– |
– |
– |
20.5 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
A: Absent background lawn development;
B0: No revertant growth and reduced background lawn development;
B<<: Reduced background lawn development and revertant colony numbers below the vehicle and historical control data ranges;
B<: Reduced background lawn development and revertant colony numbers below the vehicle control data range;
SB0: No revertant growth and slightly reduced background lawn development;
SB<<: Slightly reduced background lawn development and revertant colony numbers below the vehicle and historical control data ranges;
SB<: Slightly reduced background lawn development and revertant colony numbers below the vehicle control data range;
SB#: Slightly reduced background lawn development and revertant colony numbers within the vehicle control data range; however, below the historical control data range;
SB: Slightly reduced background lawn development and revertant colony numbers within the historical control data range and within the vehicle control data range;
PP: Pinpoint colonies;
<<: Revertant colony numbers below the vehicle and historical control data ranges;
<: Revertant colony number significantly lightly below the corresponding vehicle control range; evaluated as inhibition;
‑: No inhibition
*: Revertant colony number slightly below the corresponding vehicle control range; however, within the biological variability range of the applied test system, evaluated as no inhibition
Table 2 Summary table of the results of the initial mutation test
Initial Mutation Test (Plate Incorporation Test) |
||||||||||||||||||||
Concentrations (mg/plate) |
Salmonella typhimuriumtester strains |
Escherichia coli |
||||||||||||||||||
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2uvrA |
||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||||||||||
Mean values of revertants per plate Mutation rate (MR) |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Untreated Control |
13.0 |
0.78 |
25.3 |
1.01 |
82.3 |
1.04 |
96.3 |
1.02 |
9.7 |
0.91 |
11.0 |
1.14 |
4.3 |
0.76 |
7.7 |
1.28 |
30.7 |
0.92 |
25.3 |
0.84 |
DMSO Control |
22.0 |
1.00 |
23.7 |
1.00 |
– |
– |
97.7 |
1.00 |
– |
– |
11.0 |
1.00 |
4.3 |
1.00 |
6.0 |
1.00 |
– |
– |
39.0 |
1.00 |
Ultrapure Water Control |
16.7 |
1.00 |
25.0 |
1.00 |
79.0 |
1.00 |
94.0 |
1.00 |
10.7 |
1.00 |
9.7 |
1.00 |
5.7 |
1.00 |
6.0 |
1.00 |
33.3 |
1.00 |
30.0 |
1.00 |
3200 |
1.0 |
0.06 |
7.3 |
0.29 |
10.0 |
0.13 |
4.0 |
0.04 |
1.7 |
0.16 |
5.3 |
0.55 |
0.0 |
0.00 |
0.0 |
0.00 |
9.7 |
0.29 |
13.3 |
0.44 |
1600 |
11.0 |
0.66 |
22.3 |
0.89 |
29.0 |
0.37 |
59.0 |
0.63 |
8.7 |
0.81 |
9.7 |
1.00 |
2.7 |
0.47 |
4.7 |
0.78 |
21.3 |
0.64 |
11.3 |
0.38 |
800 |
18.3 |
1.10 |
15.0 |
0.60 |
59.0 |
0.75 |
84.0 |
0.89 |
9.7 |
0.91 |
8.3 |
0.86 |
4.3 |
0.76 |
6.7 |
1.11 |
21.3 |
0.64 |
24.7 |
0.82 |
320 |
15.7 |
0.94 |
13.0 |
0.52 |
74.7 |
0.95 |
85.3 |
0.91 |
9.3 |
0.88 |
10.0 |
1.03 |
7.0 |
1.24 |
7.7 |
1.28 |
21.0 |
0.63 |
31.7 |
1.06 |
128 |
15.3 |
0.92 |
20.0 |
0.80 |
60.0 |
0.76 |
74.0 |
0.79 |
9.3 |
0.88 |
14.3 |
1.48 |
4.7 |
0.82 |
5.7 |
0.94 |
27.0 |
0.81 |
28.3 |
0.94 |
51.2 |
14.3 |
0.86 |
20.0 |
0.80 |
65.3 |
0.83 |
78.3 |
0.83 |
9.0 |
0.84 |
12.7 |
1.31 |
4.7 |
0.82 |
6.3 |
1.06 |
24.0 |
0.72 |
32.0 |
1.07 |
20.5 |
14.7 |
0.88 |
19.0 |
0.76 |
69.3 |
0.88 |
90.7 |
0.96 |
10.0 |
0.94 |
10.3 |
1.07 |
5.3 |
0.94 |
7.0 |
1.17 |
21.7 |
0.65 |
31.7 |
1.06 |
NPD (4mg/plate) |
404.0 |
18.36 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
SAZ (2mg/plate) |
– |
– |
– |
– |
567.3 |
7.18 |
– |
– |
965.3 |
90.50 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
9AA (50mg/plate) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
1835.3 |
423.54 |
– |
– |
– |
– |
– |
– |
MMS (2mL/plate) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
594.7 |
17.84 |
– |
– |
2AA (2mg/plate) |
– |
– |
1517.3 |
64.11 |
– |
– |
781.3 |
8.00 |
– |
– |
116.7 |
10.61 |
– |
– |
85.3 |
14.22 |
– |
– |
– |
– |
2AA (50mg/plate) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
207.0 |
5.31 |
MR:
Mutation
Rate; NPD: 4-Nitro-1,2-phenylenediamine;
SAZ: Sodium azide; 9AA: 9-Aminoacridine;
MMS: Methyl
methanesulfonate; 2AA:
2-aminoanthracene
Remarks: Ultrapure
water was applied as solvent of the test item and the positive control
substance: SAZ and MMS; the DMSO was applied as solvent for positive
control substances NPD, 9AA and 2AA. The mutation rate of the test item,
SAZ, MMS and untreated control is given referring to the ultrapure
water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.
Table 3 Summary table of the results of the confirmatory mutation test
Confirmatory Mutation Test (Pre-Incubation Test) |
||||||||||||||||||||
Concentrations (mg/plate) |
Salmonella typhimuriumtester strains |
Escherichia coli |
||||||||||||||||||
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2uvrA |
||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||||||||||
Mean values of revertants per plate Mutation rate (MR) |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Untreated Control |
17.3 |
0.96 |
23.3 |
0.93 |
91.0 |
1.41 |
102.3 |
1.04 |
7.7 |
0.88 |
12.0 |
1.09 |
6.3 |
0.83 |
6.3 |
1.27 |
43.3 |
1.01 |
51.3 |
0.95 |
DMSO Control |
18.0 |
1.00 |
22.3 |
1.00 |
– |
– |
93.3 |
1.00 |
– |
– |
12.7 |
1.00 |
7.3 |
1.00 |
6.0 |
1.00 |
– |
– |
51.3 |
1.00 |
Ultrapure Water Control |
18.0 |
1.00 |
25.0 |
1.00 |
64.3 |
1.00 |
98.3 |
1.00 |
8.7 |
1.00 |
11.0 |
1.00 |
7.7 |
1.00 |
5.0 |
1.00 |
43.0 |
1.00 |
54.0 |
1.00 |
3200 |
0.0 |
0.00 |
0.0 |
0.00 |
0.0 |
0.00 |
0.0 |
0.00 |
0.0 |
0.00 |
0.0 |
0.00 |
0.0 |
0.00 |
0.0 |
0.00 |
3.3 |
0.08 |
14.3 |
0.27 |
1600 |
0.0 |
0.00 |
0.0 |
0.00 |
0.0 |
0.00 |
0.0 |
0.00 |
0.0 |
0.00 |
0.0 |
0.00 |
0.0 |
0.00 |
0.7 |
0.13 |
4.7 |
0.11 |
22.0 |
0.41 |
800 |
0.3 |
0.02 |
12.3 |
0.49 |
0.0 |
0.00 |
47.7 |
0.48 |
3.3 |
0.38 |
8.3 |
0.76 |
2.0 |
0.26 |
0.0 |
0.00 |
25.7 |
0.60 |
46.7 |
0.86 |
320 |
6.7 |
0.37 |
20.7 |
0.83 |
52.7 |
0.82 |
86.3 |
0.88 |
6.3 |
0.73 |
12.0 |
1.09 |
4.3 |
0.57 |
4.7 |
0.93 |
43.3 |
1.01 |
57.7 |
1.07 |
128 |
19.7 |
1.09 |
27.7 |
1.11 |
60.7 |
0.94 |
106.0 |
1.08 |
9.3 |
1.08 |
15.0 |
1.36 |
9.3 |
1.22 |
5.7 |
1.13 |
43.7 |
1.02 |
53.0 |
0.98 |
51.2 |
20.7 |
1.15 |
27.0 |
1.08 |
73.0 |
1.13 |
98.7 |
1.00 |
9.7 |
1.12 |
8.0 |
0.73 |
9.3 |
1.22 |
7.3 |
1.47 |
48.3 |
1.12 |
58.7 |
1.09 |
20.5 |
17.7 |
0.98 |
24.3 |
0.97 |
71.3 |
1.11 |
88.0 |
0.89 |
11.3 |
1.31 |
14.3 |
1.30 |
6.3 |
0.83 |
5.0 |
1.00 |
45.7 |
1.06 |
50.7 |
0.94 |
NPD (4mg/plate) |
505.3 |
28.07 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
SAZ (2mg/plate) |
– |
– |
– |
– |
744.0 |
11.56 |
– |
– |
1424.0 |
164.31 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
9AA (50mg/plate) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
690.7 |
94.18 |
– |
– |
– |
– |
– |
– |
MMS (2mL/plate) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
1456.0 |
33.86 |
– |
– |
2AA (2mg/plate) |
– |
– |
1477.3 |
66.15 |
– |
– |
988.0 |
10.59 |
– |
– |
156.0 |
12.32 |
– |
– |
81.0 |
13.50 |
– |
– |
– |
– |
2AA (50mg/plate) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
211.3 |
4.12 |
MR:
Mutation
Rate; NPD:4-Nitro-1,2-phenylenediamine;
SAZ: Sodium azide; 9AA: 9-Aminoacridine;
MMS: Methyl
methanesulfonate; 2AA:
2-aminoanthracene
Remarks: Ultrapure
water was applied as solvent of the test item and the positive control
substance: SAZ and MMS; the DMSO was applied as solvent for positive
control substances NPD, 9AA and 2AA. The mutation rate of the test item,
SAZ, MMS and untreated control is given referring to the ultrapure
water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.
Table 4 Historical Control Values for Revertants/Plate (for the Period of 2016-2018)
|
Bacterial strains |
||||||
Historical control data of untreated control |
‑S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
Average |
18.9 |
88.7 |
10.7 |
7.9 |
28.0 |
||
SD |
2.0 |
10.5 |
0.6 |
0.7 |
3.7 |
||
Minimum |
8 |
62 |
4 |
2 |
12 |
||
Maximum |
36 |
128 |
21 |
18 |
50 |
||
+S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Average |
24.0 |
106.5 |
11.2 |
8.3 |
35.1 |
||
SD |
1.7 |
10.5 |
0.3 |
0.7 |
3.9 |
||
Minimum |
11 |
71 |
3 |
2 |
16 |
||
Maximum |
40 |
152 |
20 |
17 |
59 |
||
|
Bacterial strains |
||||||
Historical control data of DMSO control |
‑S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
Average |
17.5 |
80.6 |
10.7 |
7.5 |
25.9 |
||
SD |
1.2 |
10.6 |
0.8 |
0.7 |
2.6 |
||
Minimum |
9 |
58 |
4 |
2 |
12 |
||
Maximum |
35 |
124 |
21 |
18 |
52 |
||
+S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Average |
22.3 |
95.6 |
10.9 |
8.1 |
34.5 |
||
SD |
1.1 |
10.3 |
0.6 |
0.7 |
4.2 |
||
Minimum |
10 |
65 |
3 |
2 |
16 |
||
Maximum |
37 |
146 |
24 |
19 |
58 |
||
|
Bacterial strains |
||||||
Historical control data of Water control |
‑S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
Average |
18.6 |
87.0 |
11.1 |
7.9 |
29.8 |
||
SD |
1.9 |
11.5 |
0.8 |
0.5 |
4.7 |
||
Minimum |
10 |
60 |
3 |
2 |
13 |
||
Maximum |
31 |
120 |
24 |
17 |
55 |
||
+S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Average |
23.6 |
105.0 |
11.3 |
8.1 |
36.8 |
||
SD |
1.8 |
11.7 |
0.7 |
0.6 |
4.0 |
||
Minimum |
13 |
76 |
5 |
3 |
18 |
||
Maximum |
41 |
148 |
19 |
17 |
63 |
||
|
Bacterial strains |
||||||
Historical control data of positive controls |
‑S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
Average |
364.6 |
1095.3 |
987.7 |
564.4 |
911.6 |
||
SD |
111.2 |
197.3 |
119.3 |
73.8 |
112.8 |
||
Minimum |
182 |
543 |
409 |
137 |
361 |
||
Maximum |
844 |
2200 |
2123 |
2265 |
1995 |
||
+S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Average |
1419.2 |
1580.8 |
168.2 |
148.2 |
203.3 |
||
SD |
249.7 |
337.3 |
22.0 |
16.3 |
5.3 |
||
Minimum |
281 |
688 |
93 |
71 |
132 |
||
Maximum |
3421 |
3333 |
349 |
367 |
364 |
Applicant's summary and conclusion
- Conclusions:
- 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 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. - Executive summary:
The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.
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 tests, a preliminary concentration 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 tests and the concentration range finding test the test item was dissolved in ultrapure water (ASTM Type I). At the preparation of the test item stock solution a correction (multiplier) factor of 1.953 (1/0.512=1.953) based on the purity of 51.2% was taken into consideration. Based on the cytotoxicity and solubility results of the preliminary concentration range finding test (informatory toxicity test) and based on the recommendations in OECD 471 guideline, the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 3200; 1600; 800; 320; 128; 51.2 and 20.5 μg/plate.
In the preliminary concentration range finding test strong inhibitory effect of the test item was observed at the recommended maximum test concentration of 5000 μg/plate.
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9) throughout the study. In the initial and confirmatory mutation tests unequivocal inhibitory effect of the test item on bacterial growth was observed. The cytotoxicity was indicated by affected background lawn development (absent, reduced or slightly reduced background lawn), affected colony development (pinpoint colonies) and decreased revertant colony counts (absent revertants or revertants below the historical control data ranges and/or corresponding vehicle control data ranges). In general, 320 μg/plate (noticed following the pre-incubation procedure in S. typhimurium strains) was considered as lowest concentration showing unequivocal cytotoxicity.
The revertant colony numbers of solvent control ultrapure water (ASTM Type I) 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 (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.
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 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.
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