lysophospholipaseIUBMB 3.1.1.5

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:

18 September 2013 - 17 December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of lysophospholipase ( (EC no.618-333-0, CAS no. 9001-85-8, EC name Lysophospholipase, Enzyme class no 3.1.1.5 )

Molecular formula:

Not available

IUPAC Name:

Active enzyme protein of lysophospholipase ( (EC no.618-333-0, CAS no. 9001-85-8, EC name Lysophospholipase, Enzyme class no 3.1.1.5 )

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:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Constituent 4

Reference substance name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available.

IUPAC Name:

Inorganic salts as a 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.

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.: PPW35424
- Expiration date of the lot/batch: 01 July 2023
- Stability under test conditions: The test material is stable for at least 24 hours at room temperature
- Storage condition of test material: Frozen (-20°C)

Method

Target gene:

Histidine and tryptophan locus in the genome of five strains of bacteria.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

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 and 5000 μg/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

Positive controls:

yes

Positive control substance:

2-nitrofluorene

other: Acridine mutagen (ICR-191), 1-Methyl-3-Nitro-N-NitroGuanidine (MNNG), 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 10^9 cells/mL.
DURATION
- Exposure duration, pre-incubation: The incubation mixtures were incubated with shaking at 37 ± 1 °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 and observation of the bacterial background lawn. 0.1 L aliquots of a 10^6 dilution of each bacterial suspension were poured on to minimal glucose agar plates in duplicates. The plates were inverted and incubated at 37 ± 2°c for about 72 hours and scored.

Evaluation criteria:

The test substance was considered as positive when it has induced at least a doubling in the mean number of revertant colonies per plate compared to the appertaining solvent control in one or more of the strains, in the presence or absence of S9 mix, if this response is dose related (at least 3 doses) and reproducible. 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:

N/A.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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 can be a concentration limiting factor, but not 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

Applicant's summary and conclusion

Conclusions:

No treatments of any of the Salmonella and E. coli strains with lysophospholipase resulted in any increases in revertant numbers that meet the criteria for a positive or equivocal response.
Lysophospholipase is not mutagenic in the Ames assay in both the presence and absence of metabolic activation.

Executive summary:

Lysophospholipase batch PPW35424 was examined for mutagenic activity in the bacterial reverse mutation assay using Salmonella typhimurium strain TA1535, TAl00, TA1537, TA98 and Escherichia coli WP2uvrApKM101. Crude enzyme preparations, like the present batch of lysophospholipase contain the free amino acids histidine and tryptophan, most often in an amount, which exceeds the critical concentration for incorporation in the direct standard assay. To overcome this problem all strains were exposed to lysophospholipase in liquid culture ("treat and plate assay"). Bacteria were exposed to 6 doses (156, 313, 625, 1250 and 5000 μg/mL dry matter) of the test substance in a phosphate buffered nutrient broth. After incubation the test substance was removed by centrifugation prior to plating.

The study was conducted with and without the metabolic activation system S9 - a liver preparation from male rats, pre-treated with Aroclor 1254, and the co-factors required for mixed function oxidase activity (S9 mix). All results were confirmed by conducting two complete and independent experiments. Lysophospholipase contains an abundance of various nutrients, and composes a rich growth medium to the test bacteria. These circumstances are reflected in the present study. No toxicity of the test substance to the bacteria is observed. Growth stimulation ("feeding effect") is present in several test series especially with addition of S9 as demonstrated by increases in the viable count of exposed cultures compared to the solvent control. These conditions have no obvious influence on the revertant colony count of the Salmonella strains. No treatments of any of the S. typhimurium and E.coli strains with lysophospholipase resulted in any increases in revertant numbers that meet the criteria for a positive or equivocal response.