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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study carriet out according to to internationally accepted guidelines. No deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
9th addendum
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese guidelines: Kanpoan No.287, Eisei No.127, Heisei 09/10/31 Kikyoku No.2
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): DBPP reaction mass
- Analytical purity:99.8%
- Composition of test material, percentage of components:
* 70,4% dibutyl phenyl phosphate
* 19,1% butyl diphenyl phosphate
* 10,3% tributyl phosphate
- Lot/batch No.: 1149785
- Expiration date of the lot/batch: july 2012
- Stability in solvent: unknown
- Storage condition of test material: +2 to +8°C

Method

Target gene:
Salmonella typhimurium histidine (his)
Escherichia coli tryptophan (trp)
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:
Mammalian Microsomal Fraction S9 Mix
Test concentrations with justification for top dose:
In the pre-experiment (reported as experiment I), the concentration range of the test item was 3-5000 ug/plate.
Since toxic effects were observed, 8 concentrations were tested in experiment II and 5000 ug/plate was chosen as maximal concentration:

3 - 10 - 33 - 100 - 333 - 1000 - 2500 and 5000 ug/plate
Vehicle / solvent:
DMSO - the solvent was chosen because of its solubility properties and it relative non-toxicity to the bacteria.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
with and without metabolic activation
Positive control substance:
other: multiple positive controls - see "details on test system and conditions"
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Experiment I: plate incorporation
- Experiment II: pre-incubation

POSITIVE CONTROL SUBSTANCES:
- Without metabolic activation:
* sodium azide -- TA 1535, TA 100
* 4-nitro-o-phenylene-diamine -- TA 1537, TA 98
* methyl methane sulfonate -- WP2 uvrA
-With metabolic activation:
* 2-aminoanthracene -- all strains

DETERMINATION OF TOXICITY:
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA.
Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described for experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

NUMBER OF REPLICATIONS: for each strain and dose level including the controls, 3 plates were used.

PRECULTURES:
From the thawed ampoules of the strains 0,5 mL suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 uL ampicillin (25 ug/mL) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8g nutrient broth (MERCK, 64293 Darmstadt/Germany) and 5 g NaCl (MERCK, 64293 Darmstadt/Germany).
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37°C The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (10E08 - 10E09 cells/mL).

PLATE INCORPORATION TEST (EXPERIMENT I):
The following materials were mixed in a test tube and poured onto the selective agar plates:
* 100 uL test solution at each dose level (solvent or reference mutagen solution)
* 500 uL S9 mix (for tests with metabolic activation) or S9 mix substitution buffer (for tests without metabolic activation)
* 100 uL bacteria suspension (cfr. precultures)

PRE-INCUBATION ASSAY (EXPERIMENT II):
In the pre-incubation assay 100 uL test solution, 500 uL S9 mix / S9 mix substitution buffer and 100 uL bacterial suspension were mixed in a test tube and shaken at 37°C for 60 minutes. After pre-incubation 2,0 mL overlay agar (45°C) was added to each tube. The mixture was poured on selective agar plates.
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the lab's historical data
- the positive control substance should produce a significant increase in mutant colony frequencies

EVALUATION OF RESULTS:
A test item is conisdered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (for TA 98, TA 100 and WP2 uvrA) or thrice (for TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
/

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for TA 1537 (as from 1000 ug/plate) and TA 100 (as from 2500 ug/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

An Ames study was performed to investigate the potential of dibutyl phenyl phosphate (DBPP) reaction mass to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent exerpiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations in both experiment I and experiment II: 3 - 10 - 33 - 100 - 333 - 1000 - 2500 and 5000 ug/plate.

The plates incubated with the test item showed reduced background growth in all strains at higher concentrations.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0,5), occurred in nearly all strains with and without activation.

No substantial increase in revertant colony numbers of any of the 5 tester strains was observed following treatment with dibutyl phenyl phosphate (DBPP) reaction mass at any dose level, neither in the presence nor absence of metabolic activation mix (S9).

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.