Lyase, pectate

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:

27 January March 1999 - 19 April 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Principles of method if other than guideline:

The ‘treat and plate’ treatment method was used.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Lyase, pectate

EC Number:

232-766-9

EC Name:

Lyase, pectate

Cas Number:

9015-75-2

Molecular formula:

Not available

IUPAC Name:

Pectate lyase IUBMB 4.2.2.2

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.

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.

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.

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.: PPE 6345
- Expiration date of the lot/batch: 2008-11-30
- Stability under test conditions: Stability at ambient temperature >4 hours
- Storage condition of test material: Stored below minus 18 degrees of C in the dark.

Method

Target gene:

The study describes experiments performed to assess the effect of the test material pectate lyase in amino acid dependent strains of Salmonella typhimurium capable of detecting both induced frame-shift (TA1537 and TA98) and base-pair substitution mutations (TA1535 and TA100); and strain of Escherichia coli WP2uvrApKM101 that can detect substitution at AT to GC or in G-C pair. The test system is a reverse mutation of amino acid dependent bacterial strains.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor 1254 induced Sprague Dawley rats

Test concentrations with justification for top dose:

Six doses 156, 313, 625, 1250, 2500 and 5000 μg/mL were tested.

Vehicle / solvent:

- Vehicle used: DI water
- Justification for choice of solvent/vehicle: The test substance is water-soluble and any human exposure will be in aqueous solutions.
- Solvent for positive controls: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene, N-Methyl-N-Nitro-NitrosoGuanidine

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 2 x 10^9 cells/mL

DURATION
- Preincubation period: 3 hrs (liquid culture assay).
- Exposure duration: Same as preincubation for treat and plate
- Incubation time (selective incubation) : 64 hrs

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count

Evaluation criteria:

According to the guideline.

Statistics:

N/A

Results and discussion

Test results

Key result

Species / strain:

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:

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
- Definition of acceptable cells for analysis: Viability and gene type control

HISTORICAL CONTROL DATA
Not speficied

Applicant's summary and conclusion

Conclusions:

Pectate lyase, batch PPE 6345, was not mutagenic when tested under the conditions applied in this bacterial reverse mutation test.

Executive summary:

Pectate lyase was tested in two independent experiments. The ‘treat and plate’ treatment method was used in each test to avoid the possibility that bio-available histidine in the test item might compromise the test. Six dose levels (156, 313, 625, 1250, 2500 and 5000 μg dry matter/mL) were tested. Pectate lyase was diluted in DI water was added to bacteria growth medium. The bacteria was also treated with the positive controls, respectively. After 3 hr treatment were bacteria washed, plated and incubated for 64 hours. The treatments were performed both in the absence and the presence of metabolic activation system (S-9 mix).

The test item was considered not toxic to the test bacteria, either in the absence or presence of S-9 mix. No increases over 2 -fold in the number of revertant colonies were observed in either experiment.

The results obtained with the solvent and positive controls demonstrated the sensitivity of the tests and the efficacy of the S-9 mix metabolic activation system.

Based on the results obtained in this study, it is concluded that pectate lyase, batch PPE 6345 was not mutagenic when tested under the conditions applied in this bacterial reverse mutation test.