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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: other: Chromosome aberration/clastogenicity and aneuploidy
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Jul 2008 - 12 Dec 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Draft OECD guideline 487, adopted 22 July 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): Catalase, PPX 28086
- Substance type: UVCB
- Physical state: liquid
- Lot/batch No.: PPX 28086
- Expiration date of the lot/batch: Stable at least until 08 April 2018
- Stability under test conditions: The undiluted test material and dilutions in water (10% and 33%) are stable for at least 24 hours at room temperature or 4 degrees Celcius
- Storage condition of test material: minus 18 degrees of C

Method

Species / strain
Species / strain / cell type:
lymphocytes: Primary cells from human blood
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Highest concentration tested was 5000 µg test substance/mL (stock solution 50 mg/mL weighed out as received) and dilutions hereof.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Purified water
- Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: Mitomycin C (MMC) and Vinblastine (VIN) in the absence of rat liver S-9, Cyclophosphamide (CPA) in the presence of S-9.
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into a 8.1mL HEPES-buffered RPMI medium containing 20% (v/v) heat inactivated foetal calf serum and 50 ug/mL gentomycin. The mitogen, phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37 ± 1°C for approximately 48 hours and rocked continuously before treatment with test article.
Sets of duplicate cultures were exposed to the test substance for 3 hours in the absence and presence of metabolic activation (S-9 mix) and harvested 24 hours after the beginning of treatment (3+21 hour treatment). Additionally, a continuous 24-hour treatment without S-9 mix was included with harvesting at the end of treatment (24+0 hour treatment).
For removal of the test article, cells were pelleted (approximately 300 g, 10 minutes), washed twice with sterile saline (pre-warmed in an incubator set to 37 ± 1°C), and re-suspended in fresh pre-warmed medium containing foetal calf serum andgentamycin. Cytochalasin-B (at a final concentration of 6 μg/mL per culture) was added to post wash off culture medium to block cytokinesis.
Three concentrations, covering an appropriate range of cytotoxicity, were selected for scoring of micronuclei by evaluating the effect of the test substance on the replication index. Where possible, 2000 cells per concentration (500 cells from each replicate culture, 1000 cells per culture) were scored.

DURATION
- pre-incubation after PHA stimulation: 48 hours
- Exposure duration: 3 (+21 recovery; +/-S-9 treatments) and 24 (+0 recovery; -S-9 treatments) hours

NUMBER OF REPLICATIONS: Sets of duplicate cultures were exposed to the test substance, in at least two independent experiments.

DETERMINATION OF CYTOTOXICITY
- Method: 5000 μg/mL was determined as max dose following a preliminary cytotoxicity Range-Finder Experiment. Cytotoxicity (%) was expressed as (100 – Relative replication Index (RI)). The highest concentration for micronucleus analysis should typically be one at which approximately 55±5% reduction in RI has occurred or should be the highest concentration tested.

Evaluation criteria:
For valid data, the test article will be considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed.
2. An incidence of cells with micronuclei at such a concentration that exceeds the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of cells with micronuclei was observed.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.

The assay will be considered valid if all the following criteria are met:
1. The binomial dispersion test demonstrates acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures, particularly where no positive responses are seen.
2. The frequency of cells with micronuclei in vehicle controls falls within the historical vehicle control (normal) ranges.
3. The positive control chemicals induce statistically significant increases in the proportion of cells with micronuclei. Both replicate cultures at the positive control concentration analysed under each treatment condition should demonstrate MNBN cell frequencies that clearly exceed the current historical vehicle control ranges.
4. A minimum of 50% of cells have gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest.
Statistics:
The proportion of MNBN cells for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test. Probability values of p equal or less than 0.05 were accepted as significant.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: from human blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH of test material is 9.0
- Effects of osmolality: no marked changes in osmolality (Shifts greated then 50mOsm/kg) or pH (shifts greater than 1 pH unit) were observed
- Evaporation from medium: no
- Water solubility: yes
- Precipitation: no
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: Preliminary cytotoxicity range-finder performed

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and positive controls were within the historical negative control ranges.



Applicant's summary and conclusion

Conclusions:
It is concluded that Catalase PPX 28086 did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of an aroclor induced rat liver metabolic activation system (S-9). Maximum concentrations analysed were either up to the recommended regulatory maximum of 5000 μg/mL (3+21 hour +/-S-9 treatments), or to 3000 μg/mL (24+24 hour –S-9 treatment), limited by toxicity, in accordance with current regulatory guidelines for the in vitro micronucleus assay.
Executive summary:

The clastogenic and aneugenic activity of catalase was investigated in cultured human peripheral blood lymphocytes by effects on the frequency of micronuclei. Division of the lymphocytes was stimulated by adding phytohaemagglutinin to the cultures. Sets of duplicate cultures were exposed to the test substance for 3 hours in the absence and presence of metabolic activation (S-9 mix) and harvested 24 hours after the beginning of treatment (3+21 hour treatment). Additionally, a continuous 24-hour treatment in absence of S-9 mix was included with harvesting 48 hours after the beginning of treatment (24+24 hour treatment). The cultures were treated with cytochalasin-B after removal of the test substance to block cytokinesis. Three concentrations, covering an appropriate range of cytotoxicity, were selected for scoring of micronuclei. A minimum of 1000 cells per concentration were scored.

The proportion of binucleate cells with micronuclei in all cultures of the vehicle controls (purified water) was within the limits of the historical ranges. The positive controls induced statistically significant increases in the proportion of cells with micronuclei, demonstrating the sensitivity of the test procedure and the metabolic activity of the S-9 mix employed.

Treatment of cells with Catalase, PPX 28086 in the absence and presence of S-9 resulted in frequencies of MNBN cells which were generally similar to, and not significantly (p ≤ 0.05) higher than those observed in concurrent vehicle controls for the majority of concentrations analysed (all treatments). Exceptions (3000 μg/mL, 3+21 hour +S-9 treatment) and (500.0 μg/mL, 24+24 hour -S-9 treatment) where small but statistically significant increases were noted, were not considered of biological importance.

It is concluded that Catalase, PPX 28086 did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of an aroclor induced rat liver metabolic activation system (S-9). Maximum concentrations analysed were either up to the recommended regulatory maximum of 5000 μg/mL (3+21 hour +/-S-9 treatments), or to 3000 μg/mL (24+24 hour –S-9 treatment), limited by toxicity, in accordance with current regulatory guidelines for the in vitro micronucleus assay.