Peroxidase

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Peroxidase has been investigated in two in vitro test systems, the Ames test and the in vitro chromosome aberration test. All tests have been performed according to current OECD guidelines, and in compliance with GLP. No evidence for genetic toxicity was observed.

Link to relevant study records

Reference

Reference 1

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)

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

Target gene:

Histidine locus in 4 strains of bacteria.

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.

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

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 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 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:

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".

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 August 1996 to 23 June 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Version from May 1983.

GLP compliance:

yes

Type of assay:

other: In-vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Remarks:

primary cultures from non-smokers

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

Experiment 1: 98.87, 141.2, 201.8, 288.2, 411.8, 588.2, 840.4, 1201, 1715, 2450, 3500, 5000 μg/mL (highest dose equivalent to 4885 μg enzyme concentrate dry matter/mL)
Experiment 2: 66.82, 89.09, 118.8, 158.4, 211.2, 281.6, 375.4, 500.6, 667.4, 889.9, 1187, 1582, 2109, 2813, 3750, 5000 μg/mL (highest dose equivalent to 4885 μg enzyme concentrate dry matter/mL)
The top concentration is the maximum concentration from the manufacturing of the enzyme.

Vehicle / solvent:

Sterile anhydrous analytical grade dimethyl sulphoxide.

Untreated negative controls:

yes

Remarks:

Sterile purified water

Negative solvent / vehicle controls:

yes

Remarks:

Sterile anhydrous analytical grade dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

Two healthy, non-smoking volunteers (male in Experiment 1 and female in Experiment 2) were used in this study. Neither donor was suspected of any virus infection nor had been exposed to high levels of radiation or hazardous chemicals.
For each experiment an appropriate volume of whole blood was drawn from the peripheral circulation on the day of culture or one day prior to culture (Experiments 1 and 2 respectively). Blood was stored refrigerated until use.
Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The highest dose level used was 5000 μg/mL. In Experiment 1, treatment in the absence of S-9 was continuous for 20 hours (20+0). Treatment in the presence of S-9 was for 3 hours only followed by a 17-hour recovery period prior to harvest (3+17). The test article dose levels for chromosome analysis were selected by evaluating the effect of Peroxidase on mitotic index. Chromosome aberrations were analysed at three consecutive dose levels. These treatment regimes were repeated in Experiment 2 and a delayed sampling time included (44+0, 3+41). Chromosome aberrations were analysed in cells receiving 20+0 hour treatments in the absence of S-9 and 3+17 hour treatments in its presence at three consecutive dose levels. The highest concentrations chosen for analysis were, 889.9 and 2813 μg/mL, in the absence and presence of S-9 respectively. The effects of single concentrations only, 500.6 and 2813 μg/mL (without and with S-9) were investigated at the delayed (44+0, 3+41) sampling time.
Appropriate negative (solvent) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges.
4-Nitroquinoline-1-oxide and cyclophosphamide were employed as positive control chemicals in the absence and presence ofliver S-9 respectively. Cells receiving these were sampled in each experiment, 20 hours after the start of treatment.

METHOD OF APPLICATION: In medium.
Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 ml heparinised blood into 9.0 ml Hepes-buffered RPMI medium containing 20% (v/v) foetal calf serum and 50 μg/ml gentamycin. Phytohaemagglutinin (PHA, reagent grade) was included at a concentration of approximately 10 μg per mL of culture to stimulate the lymphocytes to divide.

DURATION
- Preincubation period: Blood cultures were incubated for approximately 48 hours at 37°C and rocked continuously.
- Exposure duration: In Experiment 1, treatment in the absence of S-9 was continuous for 20 hours (20+0). Treatment in the presence of S-9 was for 3 hours only followed by a 17-hour recovery period prior to harvest (3+17). These treatment regimes were repeated in Experiment 2 and a delayed sampling time included (44+0, 3+41).
- Expression time (cells in growth medium): Approximately 20 hours.

STAIN (for cytogenetic assays): Giemsa in pH 6.8 Gurr’s buffer .

NUMBER OF REPLICATIONS: One set of quadruplicate cultures {A, B, C and D) for each of the treatment regimes was then treated with the solvent and one set of duplicate cultures with the test article.

NUMBER OF CELLS EVALUATED: 100 cells scored for A and B, respectively.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

Evaluation criteria:

The test article was to be considered as positive in this assay if:
1) a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurred at one or more concentrations, and
2) the proportion of cells with structural aberrations at such doses exceeded the normal range, and
3) the results were confirmed in the second experiment.

Statistics:

The frequency of cells with structural aberrations excluding gaps were compared with the vehicle control value using Fisher's exact test. Probability values of p ≤0.05 were accepted as significant.

Key result

Species / strain:

lymphocytes: human primary culture

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
- Effects of osmolality: Peroxidase had a negligible effect on osmolality.
- Water solubility: Enzymes are water soluble.
- Precipitation: Precipitation was be a confounding factor, but it is not an issue in this test.
- Definition of acceptable cells for analysis: At least 160 cells out of an intended 200 were analysable at each dose level.

HISTORICAL CONTROL DATA
- Negative (solvent/vehicle) historical control data: The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges.

Conclusions:

It is concluded that peroxidase did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its limit of toxicity in the absence and presence of S-9.

Executive summary:

Peroxidase was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures from a male and female donor in two independent experiments. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The highest dose level used was 5000 μg/mL (equivalent to 4885 μg enzyme concentrate dry matter/mL).

 

In Experiment 1, treatment in the absence of S-9 was continuous for 20 hours (20+0). Treatment in the presence of S-9 was for 3 hours only followed by a 17-hour recovery period prior to harvest (3+17). The test article dose levels for chromosome analysis were selected by evaluating the effect of Peroxidase on mitotic index. Chromosome aberrations were analysed at three consecutive dose levels. The highest concentrations chosen for analysis, 588.2 and 5000 μg/mL, induced approximately 49% and 48% mitotic inhibition (reduction in mitotic index) in the absence and presence of S-9, respectively. These treatment regimes were repeated in Experiment 2 and a delayed sampling time included (44 +0, 3 +41). Chromosome aberrations were analysed in cells receiving 20 +0 hour treatments in the absence of S-9 and 3 +17 hour treatments in its presence at three consecutive dose levels. The highest concentrations chosen for analysis were, 889.9 and 2813 μg/mL, in the absence and presence of S-9, respectively, which induced approximately 60% mitotic inhibition. The effects of single concentrations only, 500.6 and 2813 μg/mL (without and with S-9) were investigated at the delayed (44+0, 3+41) sampling time.

 

Appropriate negative (solvent) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges. 4-Nitroquinoline-1-oxide and cyclophosphamide were employed as positive control chemicals in the absence and presence of liver S-9, respectively. Cells receiving these were sampled in each experiment, 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations.

 

Treatment of cultures with peroxidase in both the absence and presence of S-9 resulted in frequencies of cells with aberrations which were similar to and not significantly different from those in concurrent negative controls. It is concluded that peroxidase did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its limit of toxicity in the absence and presence of S-9.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Due to the lack of genetic toxicity peroxidase is not classified.