Peroxidase

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:

10-03-1992 to 13-05-1992

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Version from May 1983. This version requires the use of at 4 least 4 bacteria strains, which were used in this test. The positive controls were different than the guideline.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of peroxidase (EC no. 232-668-6, CAS no. 9003-99-0, EC name Peroxidase, Enzyme class no. 1.11.1.7)

Molecular formula:

Not applicable, see remarks.

IUPAC Name:

Active enzyme protein of peroxidase (EC no. 232-668-6, CAS no. 9003-99-0, EC name Peroxidase, Enzyme class no. 1.11.1.7)

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:

solid: crystalline

Details on test material:

- Lot/batch No.: PPX 3806
- Expiration date of the lot/batch: 04 March 1994

Method

Target gene:

Histidine locus in 4 strains of bacteria.

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

1. test: 10, 3.3, 1.0, 0.33, 0.10 and 0.03 mg enzyme concentrate dry matter/mL incubation mixture.
2. test: 10, 5, 2.5, 1.25, 0.63 and 0.31 mg enzyme concentrate dry matter/mL.

Vehicle / solvent:

Distilled water used. The enzyme is water soluble.
The positive controls were dissolved in DMSO.

Controls

Untreated negative controls:

yes

Remarks:

sterile deionized water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: 2-Aminoanthracene and 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).
A liquid culture assay was applied. Samples of each strain were grown up in nutrient broth (25 g Oxoid Nutrient broth No. 2 and 5 g NaCl per liter), 16 h in a 37°C waterbath with shaking. Fresh cultures were prepared before each test. Vogel-Bonner medium agar plates with 2% glucose were prepared. All plates were stored at 4°C in closed plastic, bags and examined for contamination and dryness before use.

DURATION
- Preincubation period: 3 hours
- Exposure duration: 3 hours. Same as preincubation for treat and plate.
- Expression time (cells in growth medium): 48-72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: Viable cell count and observation of the bacterial background lawn. 0. 1 ml aliquots of a 10·5 dilution of each bacterial suspension were poured on to Nutrient agar plates. All plates with exposed bacteria were in triplicates and the negative control in 5 plates.

Evaluation criteria:

For Salmonella typhimurium strain TA 1535, TA 1537 and TA 98 at least a doubling of the mean control value and a dose related response was looked for. At high dose levels this may be reverted because of toxicity to the bacteria. For TA 100 a 50%
reproducible increase over control value is considered as indicative of a mutagenic effect.

Statistics:

None

Results and discussion

Test results

Test results 1

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 2

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 3

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 4

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

Additional information on results:

Peroxidase is a crude enzyme preparation. It contains an abundance of various nutrients, and composes a growth medium to the test bacteria. This means, that comparison of viable counts between exposed cultures and control culture reflects growth stimulation/ inhibition as well as cell killing. From the viable count it can be stated, that the test substance has no significant effect on bacterial viabilities.
It was concluded, that there was no indication of mutagenic activity of peroxidase (Batch No. PPX 3806) in the presence or absence of metabolic activation.

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Yes
- Precipitation: Precipitation is a concentration limiting factor, but it was not an issue in this test.
- Definition of acceptable cells for analysis: Viability and gene type control

HISTORICAL CONTROL DATA
- Positive historical control data: The results obtained with the positive control groups were within the normal ranges experienced in the laboratory, when a liquid culture assay is applied.
- Negative (solvent/vehicle) historical control data: All negative control values presented in this report are within the normal ranges experienced in the laboratory and reported in the literature with these strains of Salmonella typhimurium. It should be noticed, that in this "treat and plate" assay washed cultures of bacteria is added to the minimal glucose agar plates. This condition often results in slightly lower control levels than in the "plate incorporation assay".

Applicant's summary and conclusion

Conclusions:

The results of the bacterial mutagenicity tests gave no indication of the presence of mutagenic components in this preparation of peroxidase (Batch No. PPX 3806) when tested in the presence or absence of the S-9 metabolic system.

Executive summary:

Peroxidase (Batch No. PPX 3806) was examined for mutagenic activity using Salmonella typhimurium strain TA 1535, TA 100, TA 1537 and TA 98. A liquid culture assay was applied. Bacteria were exposed to 6 doses of the test substance in a phosphate buffered nutrient broth for 3 hours. After incubation the test substance was removed by centrifugation prior to plating. The number of revertants to prototrophy and viable cells were estimated.

The test was conducted in the presence and absence of metabolic activation - a liver preparation from male rats, pre-treated with Aroclor 1254, and the co-factors required for mixed function oxidase activity (S-9 mix). The sensitivity of the individual bacterial strains was confirmed by significant increases in number of revertant colonies induced in similar conditions by diagnostic mutagens.

All results were confirmed by conducting two independent experiments. No dose-related and reproducible increases in revertants to prototrophy were obtained with any of the bacterial strains exposed to peroxidase either in the presence or absence of S9-mix.

It was concluded, that the results of the experiments, described in this report, gave no indication of mutagenic activity of peroxidase (Batch No. PPX 3806) in the presence or absence of metabolic activation.