Catalase

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The present test substance, catalase IUB 1.11.1.6, has been investigated in three in vitro test systems, the Ames test, the in vitro micronucleus test and the chromosome aberration assay. All tests have been performed according to current OECD guidelines, and in compliance with GLP. No evidence for genetic toxicity was observed. These results are supported by read-across from three in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on three other enzymes, with a similar genotoxicity profile.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

other information

Justification for type of information:

According to the ECHA Guidance Chapter R 7a: Endpoint specific guidance (version 2, Nov 2012), the following studies on genetic toxicity are required: In vitro gene mutation study in bacteria and one of the following, in vitro cytogenicity study in mammalian cells or an in vitro micronucleus study. In case these studies are both negative, an in vitro gene mutation study in mammalian cells is requested in addition. The present test substance, catalase, has been investigated in three in vitro test systems, the Ames test, the micronucleus test and the in vitro chromosome aberration test. The tests have been performed according to current OECD guidelines, and in compliance with GLP. No evidence for genetic toxicitywas observed. The results are supported by read-across from three in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on three different amylases . The safety of the production strain is fully documented to belong to a safe strain lineage (Pariza and Johnson, 2001; Enzymes REACH Consortium, 2009) and the enzyme test material was well characterized. All enzyme classes are hydrophilic and readily biodegradable and in general, non-protease enzymes exhibit the same toxicological properties and although they are potential respiratory sensitizers, they are considered to be of low toxicity, confirmed by toxicity studies performed and published by the industry (summarized in Basketter et al. 2012a and 2012b). The physico-chemical properties of enzymes including logPow are very similar. They are further proteins built up of amino acids and the type, order and number of the amino acids in the polymer differs between enzymes, determining the 3-dimensional structure, the activity and specificity of the individual enzyme type. Industrial production strains typically have a history of safe use for many years in the production of technical and also often food grade enzymes. Because all enzymes are built up of the same amino acids the physical and chemical characteristics will be very similar for different enzymes, and hence read-across from other non-proteolytic enzymes (e.g. amylase) should be fully applicable. The overall conclusion is that catalase is not mutagenic and does not induce genotoxicity in the present test systems. References - Pariza, M. W., and Johnson, E. A. (2001). Evaluating the Safety of Microbial Enzyme Preparations Used in Food Processing: Update for a New Century. Regulatory Toxicology and Pharmacology, 33: 173-186. - Enzymes REACH Consortium: Safety evaluation of technical enzyme products with regards to the REACH legislation. Document from Manufacturers, Importers and/or Only Representatives of one or more enzymes, who are subject to the registration requirements pursuant to REACH, 2009. http://www.enzymes-reach.org/documents.html - D. Basketter; N. Berg; F. Kruszewski; K. Sarlo; B. Concoby. The Toxicology and Immunology of Detergent Enzymes. 2012a. J. Immunotox 9(3): 320-6. - Basketter D., Berg N., Broekhuizen C., Fieldsend M., Kirkwood S., Kluin C., Mathieu S. and Rodriguez C. Enzymes in Cleaning Products: An Overview of Toxicological Properties and Risk Assessment/Management. 2012b. Reg. Toxicol. Pharmacol, 64/1: 117-123

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Conclusions:

The conclusion is that the target substance catalase IUBMB 1.11.16 is not genotoxic.

Executive summary:

The present test substances, three different amylases as the target material catalase have been tested in in vitro gene mutation studies in L5178Y mouse lymphoma cells. All tests have been performed according to current OECD guidelines, and in compliance with GLP. No evidence for genetic toxicity was observed. This supports the conclusion that the target substance catalase is not genotoxic.

Reference 2

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

Qualifier:

according to guideline

Guideline:

other: Draft OECD guideline 487, adopted 22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

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.

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.

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.

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.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Jan. 11, 1990 - Aug. 20, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Enzymes have in general the same genotoxic profile and thus read-acroos to another enzyme class is considered fully applicable.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT (6-thioguanine resistance)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Two types of Fischer's Medium:
1) FM10 (consisted of 10% horse serum, 100 µg/ml Gentamycin and 2.5 µg/ml Fungizone)
2) FM20 (consisted of 20% horse serum, 100 µg/ml Gentamycin and 2.5 µg/ml Fungizone).
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Highest concentration tested was 5000 µg/mL (as received) and dilutions hereof.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide, benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; growth in suspension; selection phase is performed in microtitre plates

DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium): 7days
- Selection time (if incubation with a selection agent): At the end of the expression time, the culteres were counted and diluted appropriately and placed into microtitre wells. Incubation performed until scorable

SELECTION AGENT : 6-TG

NUMBER OF REPLICATIONS: Preliminary trial and two independant replicates.

DETERMINATION OF CYTOTOXICITY
- Method: Cell density by counting viable cells, expressed as relative survival

Evaluation criteria:

A test article was considered to be mutagenic if:
1) The assay was valid, and
2) Significant induced mutation (i.e. the lower 95 percentile of a treated culture exceeded the upper 95 percentile of a control culture) occurred at consecutive doses in at least one experiment, and
3) Dose-related increases in mutation could be confirmed by regression analysis in both experiments.

Statistics:

The mutation frequency was expressed as “mutants per 10E6 viable cells”. In order to calculate this, the plating efficiencies of both mutant and viable cells in the same culture were calculated. Confidence limits (95%) were assigned to mutation frequencies by using logarithmic transformation of the variances of the number of clones observed on viability and mutation plates as described by E.E. Furth et al., Anal Biochem 110: 1-8, 1981

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Maltogenic Amylase, PPY 1670, under the conditions of the test, had no mutagenic activity in cultured mouse lymphoma cells when tested to a concentration of 5000 ug/mL (expressed as test material as received) in either the absence or presence of S-9.

Executive summary:

The enzyme IUBMB 3.2.1.133, Maltogenic Amylase, PPY 1670, was assayed for its ability to induce mutation at the HGPRT locus (6-thioguanine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of three independent experiments, each conducted in the absence and presence of metabolic activation by Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9 mix).

Following a wide range of treatments, separated by half-log intervals and reaching 5000 µg/ml, cells survived all doses of Maltogenic Amylase giving relative survival values of 109% and 107% at 5000 µg/ml in the absence and in the presence of S-9, respectively. This dose together with the next 3 lower doses, were plated for viability and 6-thioguanine resistance seven days after treatment. In the second and third experiment a narrower dose range was used to maximize the chance of detection any dose related effects. The top dose plated in this experiment was again 5000 µg/ml in the absence and presence of S-9, which resulted in 95% and 124% survival respectively in experiment 2 and 103% and 96% in experiment 3.

Mutation frequencies in negative control cultures fell within normal range, and statistically significant increases in mutation were induced by the positive control chemicals 4-nitroquinoline-1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

 

In the absence of S-9 no significant increases in mutation frequency were obtained following Maltogenic Amylase treatment in experiments 1 and 3. One statistically significant result was observed at the top dose of 5000 µg/ml in experiment 2, but this was not reproducible.

 

In the presence of S-9 no significant increases in mutation frequency were obtained in experiment 1. In experiments 2 and 3, statistically significant increases in mutation frequency were obtained at intermediate dose levels, but a dose-relationship was not confirmed when analyzed by linear regression analysis. Maltogenic Amylase treatments did not therefore result in reproducible dose-related increases in mutation frequency, which would normally be required to be considered as evidence of mutation induction.

 

It was concluded that Maltogenic Amylase, under the conditions employed in this study, had no mutagenic activity in this test system.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Oct. 25, 1993 - Sept. 14, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Enzymes have in general the same genotoxic profile and thus read-acroos to another enzyme class is considered fully applicable.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT (6-thioguanine resistance)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Three types of RPMI 1640 Medium was prepared:
1) RPMI A (consisted of 0 % v/v horse serum, 100 µg/ml Gentamycin, 2.5 µg/ml Fungizone and 0.5 µg/ml Pluronic)
2) RPMI 10 (consisted of 10 % v/v horse serum, 100 µg/ml Gentamycin, 2.5 µg/ml Fungizone and 0.5 µg/ml Pluronic)
3) RPMI 30 (consisted of 20 % v/v horse serum, 100 µg/ml Gentamycin and 2.5 µg/ml Fungizone)
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Highest concentration tested was 5000 µg/mL (as received) and dilutions hereof.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- The reference chemical Monopropylene glycol (MPG) was also tested because the test chemical formulation of CTGase contains 24 % MPG.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide, benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; growth in suspension; selection phase is performed in microtitre plates

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 2days
- Selection time (if incubation with a selection agent): At the end of the expression time, the culteres were counted and diluted appropriately and placed into microtitre wells. Incubation performed until scorable

SELECTION AGENT : 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Preliminary trial and two independant replicates.

DETERMINATION OF CYTOTOXICITY
- Method: Cell density by counting viable cells using background illumination, expressed as relative survival

Evaluation criteria:

A test article was considered to be mutagenic if:
1) The assay was valid, and
2) The mutation frequency at 1 or more doses was significantly greater than that of the negative control.
3) There was a significant dose-relationship as indicated by the linear trend analysis
4) The effects described above were reproducible.

Statistics:

The mutation frequency was expressed as “mutants per 10E6 viable cells”. In order to calculate this, the plating efficiencies of both mutant and viable cells in the same culture were calculated.

Statistical significance of mutant frequencies (total wells with clones) was carried out according to the UKEMS guideline (Robison et al. (1990), In Statistical Evaluation of Mutagenicity Test Data, Cambridge University Press, pp. 102-140). Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment dose, and secondly the data were checked for a linear trend in mutant frequency with treatment dose. There tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

No statistically significant increases in mutant frequency were observed following treatment with MPG at any dose level as well.

Conclusions:

The amylase CGTase, PPA 4357, IUBMB 3.2.1.1, under the conditions of the test, had no mutagenic activity in cultured mouse lymphoma cells when tested to a concentration of 5000 ug/mL (expressed as test material as received) in either the absence or presence of S-9.

Executive summary:

CGTase, PPA 4357 was assayed for its ability to induce mutation at the tk locus in mouse lymphoma cells using a fluctuation protocol. The study consisted of a preliminary experiment and cytotoxicity range-finder experiments followed by 2 independent experiments each conducted in the presence and absence of the S-9 mix. The preliminary experiment established that CGTase did not inactivate the enzymes of S-9 mix and therefore it could be tested as supplied.

 

In the cytotoxicity range-finder experiments 6 doses of CGTase were tested, separated by 2-fold intervals and ranging from 156.25 to 5000 µg/ml. The top dose of CGTase tested yielded 36.1% and 109.6% relative survival in the absence and presence of S-9.

 

Accordingly, 5 doses of CGTase were chosen for the first experiment, separated by 2-fold intervals and ranging from 312.5 to 5000µg/ml. Four doses were plated for viability and 5-trifluorothymidine resistance 2 days after treatment. The top dose plated 5000 µg/ml yielded 91.8% and 90.6% relative survival in the absence and presence of S-9, respectively. In the second experiment 5000 µg/ml CGTase was retained as the top dose but the dose range was modified slightly. The top dose tested in this experiment yielded relative survival values of 95.7% in the absence of S-9 and 116.3% in the presence of S-9.

 

Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals. Therefore the study was accepted as valid.

 

No statistical significant increases in mutant frequency were observed following treatment with CGTase at any dose level either in absence or presence of S-9 in the two experiments.

 

It is concluded that, under the conditions employed in this study, that the tested amylase CGTase PPA 4357 is not mutagenic in this test system.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Jun. 14, 1989 - Oct. 10, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The results from the present study are used to support the evaluation of the test substance cellulase IUB 3.2.1.4 by read-across from the in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on one amylase.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT (6-thioguanine resistance)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Two types of Fischer's Medium:
1) FM10 (consisted of 10% horse serum, 100 µg/ml Gentamycin and 2.5 µg/ml Fungizone)
2) FM20 (consisted of 20% horse serum, 100 µg/ml Gentamycin and 2.5 µg/ml Fungizone).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Highest concentration tested was 5000 µg/mL (as received) and dilutions hereof.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide, benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; growth in suspension; selection phase is performed in microtitre plates.

DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium): 7 or 8 days
- Selection time (if incubation with a selection agent): At the end of the expression time, the cultures were counted and diluted appropriately and placed into microtitre wells. Incubation performed until scorable.

SELECTION AGENT: 6-TG

DETERMINATION OF CYTOTOXICITY
- Method: Cell density by counting viable cells, expressed as relative survival

Evaluation criteria:

A test article was considered to be mutagenic if:
1) The assay was valid, and
2) Significant induced mutation (i.e. the lower 95 percentile of a treated culture exceeded the upper 95 percentile of a control culture) occurred at consecutive doses in at least one experiment, and
3) Dose-related increases in mutation could be confirmed by regression analysis in both experiments.

Statistics:

The mutation frequency was expressed as “mutants per 10E6 viable cells”. In order to calculate this, the plating efficiencies of both mutant and viable cells in the same culture were calculated. Confidence limits (95%) were assigned to mutation frequencies by using logarithmic transformation of the variances of the number of clones observed on viability and mutation plates as described by E.E. Furth et al., Anal Biochem 110: 1-8, 1981

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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

COMPARISON WITH HISTORICAL CONTROL DATA:
Cells treated with the test substance, either in the absence and presence of S-9, had similar mutation frequencies as those observed in concurrent solvent controls. The negative controls were within the historical negative control ranges.

Conclusions:

The test substance, amylase batch no. PPY2693, under the conditions of the test, had no mutagenic activity in cultured mouse lymphoma cells when tested to a concentration of 5000 ug/mL (provided in test material as received) in either the absence or presence of S-9.

Executive summary:

The amylase (IUBMB 3.2.1.1) BS-G-Amylase, batch PPY 2693 was assayed for its ability to induce mutation at the HGPRT locus (6-thioguanine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of two independent experiments, each conducted in the absence and presence of metabolic activation by Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9 mix).

Following a wide range of treatments, separated by half log intervals and reaching 5000 µg/ml (tested as recived), cultures surviving the top dose of 5000 µg/ml in the absence and in the presence of S-9 showed 55% and 53% survival respectively. These, together with the next 3 lower doses, were plated for viability and 6-thioguanine resistance eight (treatments in the absence of S-9) or seven (treatments in the presence of S-9) days after treatment. In the second experiment a narrower dose range was used to maximize the chance of detection any dose related effects. The top dose plated in this experiment was again 5000 µg/ml in the absence and presence of S-9, which resulted in 50% and 117% survival respectively.

Mutation frequencies in negative control cultures fell within normal range, and statistically significant increases in mutation were induced by the positive control chemicals 4-nitroquinoline-1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

The test substance failed to induce mutation at the HGPRT locus of L5178Y mouse lymphoma cells in two independent experiments when tested to a concentration of 5000 µg/ml in the absence and in the presence of S-9. Hence, it was concluded that this amylase, under the conditions employed in this study, had no mutagenic activity in this test system.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 10, 2014 to 14 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The study describes experiments performed to assess the effect of Catalase in amino acid dependent strains of Salmonella typhimurium and Escherichia coli capable of detecting both induced frame-shift and base-pair substitution mutations. Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens.that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations.

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:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

Initial toxicity-mutation assay: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (equivalent to 1.93 to 6443 µg TOS/plate).
Confirmatory mutagenicity assay: 50, 150, 500, 1500 and 5000 μg/plate ( (equivalent to 64.4 to 6443 µg TOS/plate).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: The test substance is water-soluble and any human exposure will be in aqueous solutions.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene (for TA98, TA100, TA1535, TA1537, WP2 uvrA with S9 activation) and ICR-191 (for TA1537 without S9 activation)

Details on test system and experimental conditions:

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA. Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock into a vessel, containing 30 to 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and incubating at 37±2°C for 11 to 15 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

METHOD OF APPLICATION: treat and plate modification of the preincubation methodology (plate incorporation methodology was used only for the positive control, 2-aminoanthracene (2AA), with E. coli in the presence of S9 activation)

Treat and Plate Method: In the treat and plate method, the volumes of S9 mix, Sham mix, bacteria and test article, vehicle or positive control were increased by a factor of 2.5 or 3.5 to ensure sufficient volume of resuspended bacteria to plate the desired number of replicates. In the initial toxicity-mutation assay, a 1.25 mL aliquot of S9 mix or Sham mix was added to a tube pre-heated to 37±2°C. To this tube, 250 μL of appropriate tester strain (cells seeded) and 125 μL of vehicle, test article dilution or positive control dilution were added. After the required components were added, the mixture was vortexed and incubated for 60±2 minutes at 37±2°C with shaking. The contents of the tube were then centrifuged at 1500 x g for 10 minutes. The supernatant was removed, leaving approximately 1/10 of the bacterial cell aliquot in the tube, so as not to disturb the pellet, and the tester strain was resuspended in 0.9% saline at a volume equal to the aliquot of bacteria used above. A 110 μL aliquot of the resuspended bacteria was added to 2.5 mL of molten selective top agar (1X top agar with sterile water) at 45±2°C. In the confirmatory mutagenicity assays, a 1.75 mL aliquot of S9 mix or Sham mix was added to a tube pre-heated to 37±2°C. To this tube, 350 μL of appropriate tester strain (cells seeded) and 175 μL of vehicle, test article dilution or positive control dilution were added. After the required components were added, the mixture was vortexed and incubated for 60±2 minutes at 37±2°C with shaking. The contents of the tube were then centrifuged at 1500 x g for 10 minutes. The supernatant was removed, leaving approximately 1/10 of the bacterial cell aliquot in the tube so as not to disturb the pellet, and the tester strain was resuspended in 0.9% saline at a volume equal to the aliquot of bacteria used above. A 110 μL aliquot of the resuspended bacteria was added to 2.5 mL of molten selective top agar (1X top agar with sterile water) at 45±2°C. Following the treatment phase in each assay, each tube was plated to either two or three replicate plates based on the appropriate number of replicates for the assay. The mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37 ± 2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

Plate Incorporation Method: The plate incorporation method was used to plate the positive control (2AA) for tester strain WP2 uvrA in the presence of S9 activation. One-half (0.5) milliliter of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 50 μL of positive control were added to 2.0 mL of molten selective top agar (1X top agar) at 45±2°C. The mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37 ± 2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. For the test substance to be evaluated as positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative, if it was neither positive nor equivocal.

Key result

Species / strain:

other: 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

Positive controls validity:

valid

Additional information on results:

Sterility Results: No contaminant colonies were observed on the sterility plates for the pre-study sterility assessments or the vehicle control, the test article dilutions or the S9 and Sham mixes in each assay. Colonies that appeared to be Salmonella colonies were observed on some of the sterility plates from the initial pre-study sterility assessment. Therefore, a second sterility assessment was conducted, and no colonies (either contaminants or Salmonella) were observed.

Initial Toxicity-Mutation Assay: In Experiment B1 (Initial Toxicity-Mutation Assay), the maximum dose tested was 5000 μg per plate; this dose was achieved by diluting the test article at the Sponsor-provided concentration of 167.41 mg/mL to a concentration of 100 mg/mL and using a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. The test article formed clear solutions in sterile water for injection-quality, cell culture grade water (hereafter referred to as sterile water) from 0.030 to 3.0 mg/mL and workable suspensions from 10 to 100 mg/mL. Neither precipitate nor toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

Confirmatory Mutagenicity Assay: In Experiment B2 (Confirmatory Mutagenicity Assay), no positive mutagenic responses were observed with tester strains TA98, TA100, TA1535 or WP2 uvrA in either the presence or absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor toxicity was observed. Due to confluent bacterial growth, tester strain TA1537 was not evaluated for mutagenicity but was retested in Experiment B3. In Experiment B3 (Retest of the Confirmatory Mutagenicity Assay), no positive mutagenic responses were observed with tester strain TA1537 in either the presence or absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor toxicity was observed.

Conclusions:

Under the study conditions, Catalase was not mutagenic in the Ames assay in both the presence and absence of metabolic activation.

Executive summary:

The purpose of this study was to evaluate the mutagenic potential of Catalase by measuring its ability to induce reverse mutations in Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2uvrA in the presence and absence of Aroclor-induced rat liver S9.

The assay was performed in two phases using the treat and plate modification of the preincubation method except as noted below. The plate incorporation methodology was used only for the positive control, 2-aminoanthracene (2AA), with E.coli in the presence of S9 activation. The first phase, the initial toxicity-mutation assay, was used to establish the dose range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance. All dose levels were expressed in terms of total protein (TP).

In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate; this dose was achieved by diluting the test substance at the concentration of 167.41 mg/mL to a concentration of 100 mg/mL and using a 50μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (equivalent to 1.93 to 6443 µg TOS/plate). The test substance formed clear solutions in sterile water for injection-quality, cell culture grade water (hereafter referred to as sterile water) from 0.030 to 3.0 mg/mL and workable suspensions from 10 to 100 mg/mL. Neither precipitate nor toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000μg per plate. In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with tester strains TA98, TA100, TA1535 or WP2uvrA in either the presence or absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 μg/plate (equivalent to 64.4 to 6443 µg TOS/plate). Neither precipitate nor toxicity was observed. Due to confluent bacterial growth, tester strain TA1537 was not evaluated for mutagenicity but was retested. In the retest of the confirmatory mutagenicity assay, no positive mutagenic responses were observed with tester strain TA1537 in either the presence or absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 μg/plate (equivalent to 64.4 to 6443 µg TOS/plate). Neither precipitate nor toxicity was observed.

Under the conditions of this study, Catalase did not exhibit any mutagenic responses in either the presence or absence of Aroclor-induced rat liver S9. Therefore, it was concluded to be negative in this assay.

Reference 7

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 14, 2014 to 13 October 2014

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:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

Peripheral blood lymphocytes were obtained from a healthy non-smoking 32-year-old adult male on 22 July 2014 for the preliminary toxicity assay and from the same donor on 05 August 2014 for the definitive assay. The donor had no recent history of radiotherapy, viral infection or the administration of drugs. This system has been demonstrated to be sensitive to the clastogenic activity of a variety of chemicals (Preston et al., 1981).

Peripheral blood lymphocytes were cultured in complete medium (RPMI-1640 containing 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin, 100 μg/mL streptomycin) by adding 0.6 mL heparinized blood to a centrifuge tube containing 9.4 mL of complete medium with 1% phytohemagglutinin. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 44-48 hours.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg TP/mL (equivalent to 0.644, 1.93, 6.44, 19.3, 64.4, 193.2, 644.3, 1933, 6443 µg TOS/mL)
Chromosome Aberration Test: 2500, 3500, and 5000 μg TP/mL (equivalent to 3222, 4511, 6443 µg TOS/mL)

HPBL were exposed to vehicle alone and to nine concentrations of test substance with half-log dose spacing using single cultures. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the solvent and that of the highest dose level was measured. The number of cells in mitosis per 500 cells scored was determined in order to evaluate a possible test substance effect on mitotic index. In the absence of cytotoxicity, the highest dose tested in the definitive chromosome aberration assay was the highest dose tested in the preliminary toxicity assay.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The test substance is water-soluble and any human exposure will be in aqueous solutions.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Method of application: Treatment was carried out by refeeding the cultures with complete medium for the non-activated exposure or S9 mix for the S9-activated exposure to which was added 1 mL of test substance dosing solution, or vehicle alone. In the definitive assay, positive control cultures were resuspended in complete medium for the non-activated studies, or of the S9 reaction mixture to which was added 0.1 mL of positive control in solvent.
Duration: In both assays, HPBL cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation.
Number of replications: Duplicate cultures.
Collection of Metaphase Cells: For the preliminary toxicity and the definitive assays, cells were collected 20 hours (± 30 minutes), 1.5 normal cell cycles, after initiation of treatment to ensure that the cells are analyzed in the first division metaphase. Two hours prior to harvest, Colcemid® was added to the cultures. Cells were collected by centrifugation, treated with 0.075M KCl, washed with fixative, capped and stored overnight or longer at 2-8°C or the slides were prepared immediately after harvest. To prepare slides, the cells were collected by centrifugation and if necessary, the cells were resuspended in fresh fixative. The suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with Giemsa, permanently mounted, and identified by study number, dose level, treatment condition, harvest date, activation system, test phase, and/or replicate tube design.
Scoring for Metaphase Chromosome Aberrations (Definitive Assay): The mitotic index was recorded as the percentage of cells in mitosis per 500 cells counted. Metaphase cells were examined under oil immersion. Whenever possible, a minimum of 200 metaphase spreads containing 46 centromeres from each dose level (100 per duplicate treatment) were examined and scored for chromatid-type and chromosome-type aberrations. The number of metaphase spreads that were examined and scored per duplicate culture may be reduced if the percentage of aberrant cells reaches a significant level (at least 10% determined based on historical positive control data) before 100 cells are scored. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure was not be scored as an aberration but were considered part of the incomplete exchange. Pulverized cells and severely damaged cells (counted as 10 aberrations) were also recorded. The XY vernier for each cell with a structural aberration was recorded.
OTHER EXAMINATIONS: The percentage of cells with numerical aberrations (polyploid and endoreduplicated cells) was evaluated per 100 cells analyzed for each culture (a total of 200 per dose level).
Preliminary Toxicity Test for Selection of Dose Levels
HPBL were exposed to vehicle alone and to nine concentrations of test substance with half-log dose spacing using single cultures. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the solvent and that of the highest dose level was measured. The number of cells in mitosis per 500 cells scored was determined in order to evaluate a possible test substance effect on mitotic index. In the absence of cytotoxicity, the highest dose tested in the definitive chromosome aberration assay was the highest dose tested in the preliminary toxicity assay.

Chromosome Aberration Assay
Four dose levels were tested using duplicate cultures at appropriate dose intervals based on the toxicity profile of the test substance. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. In the absence of cytotoxicity, the highest dose evaluated for chromosome aberrations was the highest dose tested in the definitive assay. Two additional dose levels were included in the evaluation

Evaluation criteria:

Toxicity induced by treatment is based upon inhibition of mitosis and was reported for the cytotoxicity and chromosome aberration portions of the study. The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.

Statistics:

Statistical analysis of the percentage of aberrant cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the vehicle control. The Cochran-Armitage test was used to measure dose-responsiveness.
The test substance was considered positive if it induced a statistically significant and dose-dependent increase the frequency of aberrant metaphases (p ≤ 0.05). If only Fisher's exact test was statistically significant without a dose-dependent increase, the result may be considered equivocal. If neither criterion is met, the results were considered to be negative.

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Preliminary Toxicity Assay: The test substance formed workable suspensions in water at concentrations ≥ 5 mg/mL, while concentrations ≤ 1.5 mg/mL were soluble in water. The test substance was soluble in treatment medium at all dose levels tested at the beginning and conclusion of the treatment period. The osmolality in treatment medium of the highest dose level tested, 5000μg/mL, was 248 mmol/kg. The osmolality of the vehicle (water) in the treatment medium was 245 mmol/kg. The osmolality of the test substance dose level in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 20%. The pH of the highest dose level of test substance in treatment medium was 7.0. Substantial toxicity (at least 50% reduction in mitotic index relative to the vehicle control) was not observed at any dose level in any of the treatment conditions. Based on the results of the preliminary toxicity test, the dose levels selected for testing in the chromosome aberration assay were selected.

 

Chromosome Aberration Assay: In the chromosome aberration assay, the test substance formed workable suspensions in water at all tested. The test substance was soluble in treatment medium at all dose levels tested at the beginning and conclusion of the treatment period. The pH of the highest dose level of test substance in treatment medium was 7.0. The findings of the cytogenetic analysis of the non-activated 4-hour exposure group: At the highest test dose level evaluated microscopically for chromosome aberrations, 5000μg/mL, mitotic inhibition was 12%, relative to the vehicle control. The dose levels selected for analysis of chromosome aberrations were 2500, 3500, and 5000μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) group (28.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test). The findings of the cytogenetic analysis of the S9-activated 4-hour exposure group: At the highest test dose level evaluated microscopically for chromosome aberrations, 5000μg/mL, mitotic inhibition was 24%, relative to the vehicle control. The dose levels selected for analysis of chromosome aberrations were 2500, 3500, and 5000μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the CP (positive control) group (17.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test). The findings of the cytogenetic analysis of the non-activated 20-hour exposure group: At the highest test dose level evaluated microscopically for chromosome aberrations, 5000μg/mL, mitotic inhibition was 46%, relative to the vehicle control. The dose levels selected for analysis of chromosome aberrations were 2500, 3500, and 5000μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) group (37.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test).

 

The results for the positive and vehicle controls indicate that all criteria for a valid assay were met. Based on these criteria, the results are justified and do not require a repeat of any portions of the study.

Conclusions:

Under the test conditions, Catalase was considered to be negative in the in vitro mammalian chromosome aberration assay in Human Peripheral Blood Lymphocytes (HPBL).

Executive summary:

The clastogenic potential of Catalase was evaluated in the chromosome aberration assay using human peripheral blood lymphocytes (HPBL) according to the OECD guideline 473 (1997) in compliance with GLP.

The preliminary toxicity test was performed to establish the dose range for testing in the cytogenetic test. In both assays, HPBL cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. Dosing formulations were adjusted for total protein content based on the concentration as supplied at 167.41 mg/mL.

In the preliminary toxicity assay, the doses tested ranged from 0.5 to 5000 μg/mL (equivalent to 0.64 to 6443 µg TOS/mL). The test substance formed workable suspensions in water at concentrations ≥ 5 mg/mL, while concentrations ≤ 1.5 mg/mL were soluble in water. The test substance was soluble in treatment medium at all dose levels tested at the beginning and conclusion of the treatment period. The osmolality in treatment medium of the highest dose level tested, 5000μg/mL, was 248 mmol/kg. The osmolality of the vehicle (water) in the treatment medium was 245 mmol/kg. The osmolality of the test substance dose level in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 20%. The pH of the highest dose level of test substance in treatment medium was 7.0. Substantial toxicity (at least 50% reduction in mitotic index relative to the vehicle control) was not observed at any dose level in any of the treatment conditions. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 1000 to 5000μg/mL for all three treatment conditions.

In the chromosome aberration assay, the test substance formed workable suspensions in water at all tested. The test substance was soluble in treatment medium at all dose levels tested at the beginning and conclusion of the treatment period. The pH of the highest dose level of test substance in treatment medium was 7.0. Substantial toxicity (at least 50% reduction in mitotic index relative to the vehicle control) was not observed at any dose level in any of the treatment conditions. Based on these findings, the doses chosen for microscopic analysis were 2500, 3500, and 5000μg/mL (equivalent to 3222, 4511, 6443 µg TOS/mL) for all three treatment conditions. The percentage of cells with structural or numerical aberrations in the test substance-treated groups was not significantly increased relative to the vehicle control at any dose level.

Under the conditions of the assay, the test substance was concluded to be negative for the induction of structural and numerical chromosome aberrations in both the non-activated and S9-activated test systems.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The genetic toxicity of catalase has been investigated in three test systems, the Ames test, the in vitro micronucleus test and chromosome aberration assay . All tests have been performed according to current OECD guidelines, and in compliance with GLP. No evidence for genetic toxicity was observed. The results are supported by read-across from three in vitro gene mutation studies in mouse lymphoma cells performed on three different amylases . The safety of the production strain is fully documented to belong to a safe strain lineage (Pariza and Johnson, 2001) and the enzyme test material was well characterized. Because enzymes are built up of the same amino acids, the physical and chemical characteristics will be very similar for different enzymes, and hence read-across from other enzymes (e.g. amylase) should be fully applicable.

In conclusion, catalase is not mutagenic and does not induce genotoxicity in any of the presented test systems.

Reference:

Pariza, M. W., and Johnson, E. A. (2001). Evaluating the Safety of Microbial Enzyme Preparations Used in Food Processing: Update for a New Century. Regulatory Toxicology and Pharmacology, 33: 173-186.

Justification for selection of genetic toxicity endpoint
No genetic toxicity of catalase could be detected in the in vitro gene mutation study in bacteria and the in vitro cytogenicity study in mammalian cells. The production strains of catalase further meet the criteria for safe strain production micro-organisms. The conclusion that catalase is non-genotoxic is supported by the read-across from in vitro gene mutation studies in mouse lymphoma cells of three other enzymes.

Justification for classification or non-classification

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