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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:
From November 8th to 25th,2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
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,1997
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tris(ethyl acetoacetato-O1',O3)aluminium
EC Number:
239-343-8
EC Name:
Tris(ethyl acetoacetato-O1',O3)aluminium
Cas Number:
15306-17-9
Molecular formula:
C18H27AlO9
IUPAC Name:
tris(ethyl acetoacetato-O1',O3)aluminium
Test material form:
solid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

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:
S9 tissue fraction prepared from livers of Sprague-Dawley rats pretreated with Phenobarbital and 5,6-benzoflavone.
Test concentrations with justification for top dose:
Concentrations used in main assays (first experiment- plate incorporation method- and second experiment- pre-incubation method): 5000, 2500,1250 ,625 and 313 µg/plate for all stains.
These concentrations were chosen based on the result on preliminary toxicity test.
Vehicle / solvent:
- Solvents used: DMSO for test item; DMSO or water for injection to dissolve positive controls.

- Justification for choice of solvent/vehicle: solubility of the test item was evaluated in a preliminary trial using sterile water for injection and DMSO. These solvents were selected since they are compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be not soluble in sterile water for injection. A suitable solution for serial dilutions was obtained at 50 mg/ml of test item in DMSO.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Remarks:
Without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9
Details on test system and experimental conditions:
BACTERIA STRAIN
Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and a strain of Escherichia coli (WP2 uvrA) were used in this study. Permanent stocks of these strains are kept at -80 °C in the laboratory. Overnight subcultures of these stocks were prepared for each day’s work. Bacteria were taken from vials of frozen cultures, which had been checked for the presence of the appropriate genetic markers, as follows:
- Histidine requirement: No Growth on Minimal plates + Biotin and Growth on Minimal plates + Biotin + Histidine.
- Tryptophan requirement: No Growth on Minimal agar plates and Growth on Minimal plates + Tryptophan.
- uvrA, uvrB: Sensitivity to UV irradiation.
- rfa: Sensitivity to Crystal Violet.
- pKM101: Resistance to Ampicillin.

MEDIA
- Nutrient Broth: Oxoid Nutrient Broth No. 2 was prepared at a concentration of 2.5% in distilled water and autoclaved prior to use. This was used for the preparation of liquid cultures of the tester strains.
- Nutrient Agar: Oxoid Nutrient Broth No. 2 (25 g) and Difco Bacto-agar (15 g) were added to distilled water(1 litre) and autoclaved. The solutions were then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
- Minimal Agar: Minimal medium agar was prepared as 1.5% Difco Bacto-agar in Vogel-Bonner Medium E, with 2% Glucose, autoclaved and poured into 9 cm plastic Petri dishes.
- Top Agar: "Top Agar" (overlay agar) was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water and autoclaved. Prior to use, 10 ml of a sterile solution of 0.5 mM Biotin + 0.5 mMHistidine (or 0.5 mM tryptophan) was added to the top agar (100 ml).

S9 TISSUE
One batch of S9 tissue fraction was used in this study and had the following characteristics:
- Species: Rat
- Strain Sprague: Dawley
- Tissue: Liver
- Inducing Agents: Phenobarbital – 5,6-Benzoflavone
The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 ml): S9 tissue fraction 1.0 ml+ NADP (100 mM) 0.4 ml+G-6-P (100 mM) 0.5 ml+ KCl (330 mM) 1.0 ml+ MgCl2 (100 mM) 0.8 ml+ Phosphate buffer (pH 7.4, 200 mM) 5.0 ml+ Distilled Water 1.3 ml

PRELIMINARY TOXICITY TEST:
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the Main Assays. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

MAIN ASSAY
Two Main Assays were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point.
In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures.
The first experiment was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation. The overlay mixture was composed as follows: Overlay agar (held at 45°C) 2.0mL, Test or control item solution 0.1ml, S9 mix or phosphate buffer (pH7.4, 0.1M) 0.5ml, Bacterial suspension 0.1ml.
The second experiment was performed using a pre-incubation method. The components were added in turn to an empty test-tube: Bacterial suspension 0.1 ml, Test item solution or control item solution 0.05 ml, S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5ml. The incubate was vortexed and placed at 37°C for 30 minutes. Two ml of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.

INCUBATION AND SCORING
The prepared plates were inverted and incubated for approximately 72 hours at 37 °C. After this period of incubation, plates were held immediately scored by counting the number of revertant colonies on each plate.

METHOD OF APPLICATION: plate incorporation in the fist experiment and pre-preincubation in the second experiment.

ACCEPTANCE CRITERIA:
The assay was considered valid if the following criteria were met:
1.Mean plate counts for untreated and positive control plates should fall within 2standard deviations of the current historical mean values.
2. The estimated numbers of viable bacteria/plate should fall in the range of100 – 500 millions for each strain.
3. No more than 5 % of the plates should be lost through contamination or other unforeseen event.




Evaluation criteria:
For the test item to be considered mutagenic ,two-fold(or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
Statistics:
The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homosce-dasticity assumptions. The regression equation is expressed as: y= a+bx

where:
y = transformed revertant numbers
a = intercept
b = slope value
x = dose level (in the units given).
The regression line does not include the untreated control data, but includes the solvent control data.
Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation co-efficient (r),the value of students "t" statistic, and the p-value for the regression lines are also given.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Slight citotoxicity observed for all tester strains at the highest concentration only with S9 in pre-incubation test.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
MAIN ASSAYS
On the basis of the results obtained in the preliminary toxicity test, in Main Assay I (Plate incorporation method) ,the test item was assayed at the following dose levels: 5000, 2500,1250,625 and 313µg/plate. No toxicity, as indicated by thinning of the background lawn and/or reductionin revertant numbers, neither increases in the number of revertant colonies were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism. As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II (pre-incubation method). The test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 µg/plate. As light toxicity,as indicated by thinning of the background lawn, was noticed with all tester strains,at the highest concentration tested, only in the presence of S9 metabolism.
No increases in revertant numbers were observed, at any dose level ,with any tester strain, in the absence or presence of S9 metabolic activation.
No precipitation of the test item was observed, at the end of the incubation period, in any experiment, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism.
The test item did not induce relevant increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism. The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

TOXICITY PRELIMINARY TEST
The test item Tris(ethylacetoacetato-O1’,O3)aluminium was assayed in the toxicity test at a maximum dose level of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. At the end of the incubation period, no precipitation of the test item was observed with any tester strain ,at any concentration tested, in the absence or presence of S9 metabolism. No toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, neither increases in the number of revertant colonies were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism.

ACCEPTANCE CRITERIA AND EVALUATION:
Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data.
The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. No plates were lost through contamination or cracking. The study was accepted as valid.
The test item did not induce two-fold increases in the number of revertant colonies, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

EVALUATION OF THE RESULTS
The test item did not induce two-fold increases in the number of revertant colonies, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Applicant's summary and conclusion

Conclusions:
The test item Tris (ethyl acetoacetato-O1’,O3) aluminium does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism.
Executive summary:

The test item Tris (ethyl acetoacetato-O1’,O3) aluminium was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with Phenobarbital and 5,6-benzoflavone.

The test item was used as a suspension/solution in dimethylsulfoxide (DMSO).

 

Toxicity test

The test item Tris (ethyl acetoacetato-O1’,O3) aluminium was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No precipitation of the test item was observed, at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. No toxicity neither increases in the number of revertant colonies were observed with any tester strain, at any dose level , in the absence or presence of S9 metabolism.

 

Main Assays

On the basis of toxicity test results ,in Main Assay I ,using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 µg/plate. No toxicity neither increases in the number of revertant colonies were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism. As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 µg/plate. A slight thinning of the background lawn was noticed with all tester strains, at the highest concentration tested, only in the presence of S9 metabolism. No increases in revertant numbers were observed, at any dose level, with any tester strain, in the absence or presence of S9 metabolic activation. At the end of the incubation period, no precipitation of the test item was observed with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism, both in Main Assay I and Main Assay II. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

 

Conclusion

It is concluded that the test item Tris (ethyl acetoacetato-O1’,O3) aluminium does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

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