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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:
04-01-2016 to 25-04-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 1997.
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Nuclease, deoxyribo-
EC Number:
232-667-0
EC Name:
Nuclease, deoxyribo-
Cas Number:
9003-98-9
Molecular formula:
n.a.
IUPAC Name:
Nuclease, deoxyribo-
Constituent 2
Reference substance name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available
IUPAC Name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 3
Reference substance name:
Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 4
Reference substance name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
Constituent 5
Reference substance name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Test material form:
liquid
Details on test material:
- Lot/batch No.: PPW38822
- Expiration date of the lot/batch: Usually 10 years from the date manufacturing.
- Storage condition of test material: -10ºC

Method

Target gene:
Histidine and tryptophan locus in the genome of five strains of bacteria
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 pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix from Aroclor 1254-induced rat liver
Test concentrations with justification for top dose:
Preliminary test: No preliminary trials were carried out.
Six concentrations of the test item (156, 313, 625, 1250, 2500, 5000 μg TOS/mL)
Vehicle / solvent:
Vehicle for enzyme: Purified water.
Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
Solvent for the positive control: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
other: Acridine mutagen (ICR-191), 2-Aminoanthracene (2-AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation in suspension, followed by plating on agar plates (treat and plate method)
- Cell density at seeding (if applicable): Overnight culture of approximately 1x 10^9 cells/mL

DURATION
- Exposure duration, pre-incubation: The incubation mixtures were incubated with shaking at 37 ± 2°C for 3 hours (treat and plate).
- Incubation time (selective incubation): about 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count.
Evaluation criteria:
The test substance is considered mutagenic when it induces at least a doubling in the mean number of revertant colonies per plate, compared to the appertaining solvent control. The response must be dose related, reproducible between replicates and experiments, and independent of growth stimulation of non-revertant bacteria. The response can be observed in one or more of the bacterial strains and in the absence or presence of S9 mix. In case of a dose related and reproducible numerical increase, which is below a doubling but at least 50% higher than the solvent control, the result is considered as equivocal and needs further clarification.
Statistics:
Not performed.

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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Positive controls validity:
valid
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
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
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Yes
- Precipitation: Precipitation is a concentration limiting factor, but no issues were reported in this study.
- Definition of acceptable cells for analysis: Viability and gene type control

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes, solvent controls.

Applicant's summary and conclusion

Conclusions:
It was concluded that Phospho-Di-Esterase (PDE), batch PPW38822 is not mutagenic in histidine-requiring Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537), and tryptophan-requiring Escherichia coli strain (WP2uvrApKM101), when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg TOS/mL with and without the rat liver metabolic activation system (S9), and using a treat and plate assay.
Executive summary:

Phosho-Di-Esterase (PDE), batch PPW38822 was examined for mutagenic activity in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium strain TA1535, TA100, TA1537, TA98 and Escherichia coli WP2uvrApKM101. Crude enzyme preparations, like the present test substance contain free amino acids histidine and tryptophan, most often in an amount, which exceed the critical concentration for incorporation in the direct standard assay (plate incorporation method). To overcome this problem, all strains were exposed to the test substance in liquid culture in a treat and plate method. Bacteria were exposed to six doses of the test substance in a phosphate buffered nutrient broth for 3 hours with 5000 μg TOS/mL as the highest concentration. After incubation, the test substance was removed by centrifugation prior to plating. The study was conducted without and with the metabolic activation system (S9 mix). The metabolic activation system was a liver preparation from male rats pre-treated with Aroclor 1254 (S9), added with co-factors required for mixed function oxidase activity. Two complete and independent experiments were conducted before any conclusions were made.

Phosho-Di-Esterase (PDE), batch PPW38822 exposure did not increase the number of revertant colonies of the Salmonella and E. coli strains, that meet the criteria for a positive or equivocal response.

Based on the results obtained in this study it was concluded that Phosho-Di-Esterase (PDE), batch PPW38822, is not mutagenic when tested under the conditions applied in this bacterial reverse mutation test.