Pullulanase

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The present test substance, pullulanase IUB 3.2.1.41, has been investigated in the following in vitro test systems, the Ames test, the in vitro chromosome aberration test, the human lymphocyte micronucleus assay and the mouse lymphoma assay. 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

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 March 2013 - 14 June 2013

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

Principles of method if other than guideline:

The ‘treat and plate’ treatment method was used in each test to avoid the possibility that bio-available histidine in the test item might compromise the test.

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

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor 1254 induced Sprague Dawley rats , was obtained crom Cappel/MP Biomedicals, LLC, and Moltox.

Test concentrations with justification for top dose:

Six doses 156, 313, 625, 1250, 2500 and 5000 μg dry matter/mL) were tested. Highest dose 1.6 mg active enzyme protein (aep)/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionized 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:

other: 2-Nitroflurene, Acridine mutagen ICR-191, 1-Methyl3-Nitro-N-NitrosoGuanidine, 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: All applications were done in medium before plating, i.e. a liquid culture assay (treat and plate assay).

DURATION
- Exposure duration: 3 hrs (liquid culture assay)
- Incubation time (selective incubation) : 64-72 hrs
DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count

Evaluation criteria:

The numbers of revertant colonies at each treatment test point were compared to the corresponding solvent control values for each set of triplicate plates.
The tests were considered to be valid as all the following criteria were met:
- negative and positive control data were consistent with the historical control data for this laboratory
- the positive control data showed marked increases over the concurrent negative control values
- the evaluation of the data was not restricted by loss of plates (e.g. through contamination).
The test item would have been considered to have shown evidence of mutagenic activity in this study if all of the following criteria had been met:
- increases in the numbers of revertant colonies were observed at one or more test points
- the mean number of revertant colonies at the test point showing the largest increase was more than twice the corresponding negative control value
- there was a credible scientific explanation for the observed dose-response relationship that involved a mutagenic effect of the test item
- the increases were reproducible between replicate plates and were observed in both main tests (when treatment conditions were the same)
- the increases were statistically significant
- the increases were not directly related to increased growth of the non-revertant bacteria

Statistics:

The numbers of revertant colonies at each treatment test point in the main tests were compared to the corresponding solvent control values. The test substance would be considered as positive if it has induced at least a doubling in the mean number of revertant colonies per plate, compared to the appertaining solvent control in one or more of the strains, in the presence or absence of S9 mix, if this response is dose related (at least 3 doses) and reproducible.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: In the first experiment, the test substance was not toxic to the bacteria: no reductions in the growth of the background lawn of non-revertant bacteria were observed in any of the test series. In the second experiment, toxicity was observed in TA100, TA1535 and TA1537 at the one to three highest dose levels (1250-5000 µg/mL), especially in the absence of S9 mix. This could be seen from reductions in the growth of the background lawn and the presence of micro-colonies of non-revertant bacteria, accompanied by reductions in the viable cell counts. The difference in toxicity between the first and second experiment, most pronounced in TA100 and TA1535 without S9 mix, could indicate a technical error in one of the experiments. Therefore, a third experiment with those two test series was included. In accordance with the first experiment, no signs of toxicity were observed in the third experiment.
On the basis of these results, 5000 ug test substance/plate was chosen as the highest dose level for the main tests.
COMPARISON WITH HISTORICAL CONTROL DATA: The solvent and positive control values were compatible with the historical control values for this laboratory.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Pullulanase, batch PPY34283, was not mutagenic when tested under the conditions applied in this bacterial reverse mutation test.

Executive summary:

Pullulanase was tested in three independent main tests. The ‘treat and plate’ treatment method was used in each test to avoid the possibility that bio-available histidine in the test item might compromise the test. Six dose levels ( 156, 313, 625, 1250, 2500 and 5000 μg dry matter/mL) were tested. Pullulanase dilluted in deionized water was added to bacteria growth medium. After 3 hr treatment were bacteria washed, plated and incubated for 64 -72 hrs. Negative control plates were treated with DI water without Pululanase. The treatments were performed both with and without a metabolic activation system (S-9 mix).

The test item was considered not toxic to the test bacteria, either in the absence or presence of S-9 mix: no reductions in the growth of the background lawn of non-revertant bacteria or the number of revertant colonies were observed were observed in two independent experiments.

No increases in the number of revertant colonies were observed at any dose level of the test item in either main test.

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

Based on the results obtained in this study, it is concluded that Pullulanase, batch PPY34283 did not show evidence of mutagenic activity when tested under the conditions applied in this bacterial reverse mutation test.

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:

February 15, 2013-August 21, 2013

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/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.

Untreated negative controls:

yes

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 sufficient volume of HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin. 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+24 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 and penicillin / streptomycin. 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. Were 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 (+24 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:

other: Minor. NMBN tested up to 5mg/mL

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH of test material is neutral, 5.6
- 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:

Pullulanase, PPY34283 did not induce micronuclei in cultured human peripheral blood lymphocytes following pulse (3+21 hour) treatments in the
absence and presence of an Aroclor induced rat liver metabolic activation system (S-9). Concentrations were tested up to 5000 μg/mL, the recommended regulatory maximum concentration for in vitro micronucleus assays.
Pullulanase, PPY34283 induced micronuclei following an extended 24+24 hour treatment in the absence of S-9 in a single experiment (at high
concentrations of 4000 and 5000 μg/mL). However, this effect was not reproduced in four further experimental trials under this treatment condition using three aliquots from the same test article batch. Similar levels of toxicity were observed in all experiments. The isolated increases seen in the single experiment were therefore non-reproducible and considered non-representative. As such, this observation was not considered of biological importance.

Executive summary:

The clastogenic and aneugenic activity of Pullulanase 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.

The 3+21 hour treatment of the cells resulted in frequencies of micronucleated binuclear cells (MNBN cells), which were similar to and not significantly (p ≤ 0.05) higher than those observed in concurrent vehicle controls.

The 24 +24 hour treatment of the cells resulted on MNBN cells, which were generally similar and not significantly higher that those observed in concurrent vehicle controls. A single experiment induced micronuclei following an extended 24+24 hour treatment in the absence of S-9 at high concentrations of 4000 and 5000 μg/mL. The observation was not repeated and therefore was not considered of biological importance.

 

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 April 2013 - 05 June 2013

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)

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at the time of study conduct.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: human peripheral blood lymphocytes (HPBL)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity assay: 0.5, 1.5, 5, 15, 50, 150, 500, 5000 μg/mL (dosing formulations adjusted for total protein content based on concentration as supplied at 76.26 mg/mL)

Chromosome aberration assay (non-activated and S-9 activated 4-hour exposures): 1500, 2450, 3500, 5000 μg/mL (dosing formulations adjusted for total protein content based on concentration as supplied at 76.26 mg/mL)

Chromosome aberration assay (non-activated 20-hour exposures): 100, 250, 500, 1000, 1250, 1500 μg/mL (dosing formulations adjusted for total protein content based on concentration as supplied at 76.26 mg/mL)

Vehicle / solvent:

Water was used as the vehicle based on the Sponsor’s request and compatibility with the target cells.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 44-48 hours
- Exposure duration: 4 hours with and without S9; 20 hours without S9
- Expression time (cells in growth medium after washing): with and without S9 - 16 hours, without S9 - 0 hours. Total time for all tests was 20 hours.
- Selection time (if incubation with a selection agent): last 2 hours of time in medium for all tests
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid®
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 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

DETERMINATION OF CYTOTOXICITY
- Method: reduction in mitotic index relative to the vehicle control

OTHER: Two hours prior to harvest, Colcemid® was added to the cultures at a final concentration of 0.1 μg/mL. Cells were collected by centrifugation, treated with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 3:1 v/v), capped and stored overnight or longer at 2-8°C. 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 the BioReliance study number, dose level, treatment condition, harvest date, activation system, test phase, and/or replicate tube design.

Evaluation criteria:

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.

A test article was considered positive if it induced a statistically significant and dose-dependent increase in the frequency of aberrant metaphases (p ≤ 0.05). If only one criterion was met (statistically significant OR dose-dependent increase), the result was considered equivocal. If neither criterion was met, the results were considered to be negative.

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.

Key result

Species / strain:

other: human peripheral blood lymphocytes (HPBL)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Substantial toxicity was observed at dose levels ≥1000 μg/mL in the non-activated 20-hour exposure group.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: HPBL cells were exposed to 9 dose levels of the test substance ranging from 0.5 to 5000 μg/mL, as well as vehicle controls, in both the absence and presence of an Aroclor-induced S9 metabolic activation system for 4 hours, or continuously for 20 hours in the absence of S9 activation. The initial preliminary toxicity assay was terminated due to contamination observed during the preparation of slides, and a repeat toxicity assay conducted using the same concentration range and test conditions as in the preliminary assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the repeat toxicity assay, no substantial toxicity (at least 50% reduction in mitotic index relative to the vehicle control) was observed at any dose level in the non-activated and S9-activated 4-hour treatment groups. Substantial toxicity was observed at dose levels≥1500 μg/mL in the non-activated 20-hour exposure group.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative

This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
The test item was considered to be negative in the In Vitro Mammalian Chromosome Aberration Test in HPBL.

Executive summary:

The test article was tested in the chromosome aberration assay using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system. A preliminary toxicity test was performed to establish the dose range for testing in the cytogenetic test. The chromosome aberration assay was used to evaluate the clastogenic potential of the test article.

Based on the findings of the preliminary toxicity test, the doses chosen for the chromosome aberration assay ranged from 1500 to 5000 μg/mL for the non-activated and S9-activated 4-hour exposure groups, and from 100 to 1500 μg/mL for the non-activated 20-hour exposure group. In the chromosome aberration assay, the 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. No substantial toxicity (at least 50% reduction in mitotic index relative to the vehicle control) was observed at any dose level in the non-activated and S9-activated 4-hour treatment groups. Substantial toxicity was observed at dose levels≥1000 μg/mL in the non-activated 20-hour exposure group. Based on these findings and upon consultation with the Sponsor, the doses chosen for microscopic analysis were 2450, 3500, and 5000 μg/mL for the non-activated and S9-activated 4-hour exposure groups, and 250, 500, and 1000 μg/mL for the nonactivated 20-hour exposure group.

The percentage of cells with structural or numerical aberrations in the test article-treated groups was not significantly increased relative to the vehicle control at any dose level (p > 0.05, Fisher's Exact test). Under the conditions of the assay described in this report, 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. The test substance was considered to be negative in the In Vitro Mammalian Chromosome Aberration Test in HPBL.

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:

key study

Study period:

26 October 1993 - 18 February 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

Thymidine kinase (tk) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fishers medium (+pen/strep, 0.1% Pluronic F68, sodium pyruvate, HEPES, 10% and 5% horse serum)
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Cells were exposed to series of test concentrations with the highest concentration tested 5000 µg/mL. Pullulanase was tested as supplied.

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium, growth suspension.
DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): Cells were allowed to grow for 2 days after exposure.
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days after the end of the expression time.

SELECTION AGENT (mutation assays): TFT (Trifluorothymidine)

NUMBER OF REPLICATIONS: one preliminary range finder and two independent replicates in the definitive mutagenesis assay

DETERMINATION OF CYTOTOXICITY
- Method: Cell density by counting viable cells, expressed as percentage relative growth in suspension (RSG%)

Evaluation criteria:

A test article was considered positive if:
- The assay was valid based on negative and positive results, and
- Response with evidence of dose-response and an induced mutation frequency of at least 70x10^-6 at relative total growth (RTG) > or = 10%

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:

It was concluded that pullulan 6-glycanohydrolase (Pullulanase) did not induce gene and chromosomal mutations at the thymidine kynase (TK) locus in the absence and presence of exogenous S9 metabolic activation, when tested up to concentration of 5000 µg/mL. Pullulanase was tested as supplied.

Executive summary:

The capability of pullulan 6-glycanohydrolase (Pullulanase) was assessed in L5178Y mouse lymphoma cells to induce gene and chromosomal mutations at the thymidine kinase (tk) locus in the absence and presence of exogenous S9 metabolic activation. Preliminary range-finding assay was followed by definitive mutagenesis assay and confirmatory mutagenesis assay.

Pullulanase was readily soluble in water, no precipitate was observed in any culture, and presence of Pullulanase had little influence on osmolality in relation to solvent controls (both with and without S9). The absolute cloning efficiencies of the medium controls in the absence and presence of metabolic activation, and the average spontaneous mutant frequencies met the criteria for acceptability. The positive control mutant frequencies were within the historical ranges for the laboratory.

Based on data in the preliminary range-dose finding study, Pullulanase was tested at concentrations up to 5000 µg/mL(as supplied) in the definitive and confirmatory mutagenesis assay, in the absence and presence of exogenous S9 metabolic activation. Pullulanase depressed % relative growth in suspension (RTG) to about 63 and 52% of solvent control, definitive and confirmatory mutagenesis assay, respectively. Pullulanase did not depress %RTG in the presence of metabolic activation, in either definitive or confirmatory mutagenesis assay.

In all tested conditions Induced Mutant Frequency >or = 70x10^-6. Pulullanase was reproducibly non-toxic and negative for the induction of gene and chromosomal mutations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The present test substance, pullulanase IUB 3.2.1.41, has been investigated in two in vivo, Klimisch-score 2 chromosome aberration studies, one in vivo mouse bone marrow chromosome aberration test and one in vivo mouse bone marrow micronucleus test. The studies were performed according to the principles of current OECD guidelines, and in compliance with GLP. No evidence for genetic toxicity was observed.

No evidence for genetic toxicity was observed and thus, it can be concluded that pullulanase is not mutagenic and does not induce chromosome aberration in the present test systems.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

October 26, 1993-August 24, 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

The OECD guideline came later (in 1997) but the overall principles of the guideline was followed in the study.
Single treatment (IP) performed, followed by 12, 24 and 48 hour sampling of bone marrow.

GLP compliance:

yes

Remarks:

incl. certificate

Type of assay:

other: micronucleus assay

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

Weight: 23 to 28g
From: Charles River Laboratories, Portage, Michigan
Randomized: removed randomly from shipping cages and randomly assigned to cages
Cages: microisolator
No. of mice/cage: 5 (sex separated)
Food: Purina Rodent Laboratory Chow No. 5002C ad libitum
Water: ad libitum
Acclimatization: 6 days before dosing
Mice identification: tail mark
Room tempreature: 19-22 degrees C
Humidity: 24-78%

Route of administration:

intraperitoneal

Vehicle:

Phosphate buffered saline (PBS)

Details on exposure:

10 mL/kg
IP to 15 males and 15 females/ Pullulanase/ dose; 5 doses in total
IP to 5 males and 5 females / positive control
IP to 5 males and 5 females / vehicle control

Duration of treatment / exposure:

Pullulanase injected mice were killed 12, 24 and 48 hours after dosing
Positive- and vehicle control mice were killed 24 hours after dosing

Frequency of treatment:

Single injection

Post exposure period:

Mice were killed 12, 24 and 48 hours after dosing.

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

889 mg/kg bw/day (nominal)

Dose / conc.:

1 581 mg/kg bw/day (nominal)

Dose / conc.:

2 811 mg/kg bw/day (nominal)

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females/dose/time point = 15/sex/dose

Control animals:

yes

Positive control(s):

0.4 mg triethylenemelamine (TEM)/kg b.w., dosed by ip injection.
TEM and the dose 0.4 mg/kg was selected because this was known to give reproducible increase in the frequency of micronucleated PCEs in Swiss Webster mice.

Tissues and cell types examined:

bone marrow from both femora

Details of tissue and slide preparation:

One 15-ml centrifuge tube containing 750 uL of bovine calf serum was prepared for each animal.
Both femora from mice killed by cervical dislocation were cleaned, and the bone marrow canal flushed with 1 mL disposable syringe with 25 G needle with 0.2 mL fetal calf serum into the centrifuge tube. Cells were centrifuged for 5 min. at 100 x g, the supernatant was decanted, pellet was mixed in the remaining fluid, and the suspended cells were placed onto slides. The drop was spread by spreader slide to even film, slide was air dried, fixed in methanol for 2 minutes, and dried.

Two slides were prepared per animal. Slides were stained for 20 min. in 5% Giemsa (in PBS with 3% methanol and 3% 0.1M citric acid), rinsed in DI water, and air dried. Slides were protected by coverslip attached with Permount, and analyzed at 100X, oil immersion, magnification.

Preliminary evaluation with uncoded slides (dose range finding):
% of PCEs (polychromatic erythrocytes) in 1000 erythrocytes from one animal per sex, from each top dose and vehicle control at 12, 24 and 48 hours after exposure were evaluated to select the highest dose. No clear depression of PCEs was detected, therefore the highest dose was selected as the highest dose scored.

Evaluation with coded slides:
Micronuclei were scored from male and female slides at 12, 24 and 48 hours after exposure to the highest dose, the next two lower doses, positive and negative controls. Slides were divided into two identical groups and coded by individual not involved in scoring. Two observers were utilized, one for each set of slides.
When slides were decoded and tabulated, the ratios of PCEs per 5000 erythocytes, and the number of micronucleated cells per 1000 PCEs and per 1000 NCEs were calculated per each treatment group.

Evaluation criteria:

Micronuclei are round and oval shaped bodies found in the cytoplasm; refractile and improperly shaped cells are not scored. Cells with more micronuclei are scored as a single micronucleated cell.

Criteria for interpretation:
Positive:
There is dose-related increase in micronuclei at at least one collection time.
And if one or more doses induces a statistically significant (p<0.05) increase in micronuclei induction.

Negative:
The positive criteria are not met.

Both biological and statistical significance are considered together.

Statistics:

No statistical analysis was required to evaluate results.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative
Pullulanase did not induce micronuclei (clastogenic effects) in male or female mice at doses up to 5000 mg Pullulanase/kg bw when injected by IP injection.

Executive summary:

In a dose range-finding study 3 male and 3 female mice/dose received vehicle (PBS 10 mL/kg), 50, 158.1, 500, 1581, or 5000 mg (half-log doses) Pullulanase/kg by ip injection. The top dose 5000mg/kg was not toxic to mice and selected as the highest dose for the micronucleus testing.

In the main study, the micronuclei from bone marrow of 15 male and 15 female mice/dose were analyzed. Mice were given one of 5 doses, 500, 889, 1581, 2811 or 5000 mg Pullulanase/kg, by ip injection. Five male and 5 female mice were exposed to vehicle or positive control. Bone marrow cells were collected from 5 male and female mice/time killed 12, 24 and 48 hours after exposure. Cells were collected by flushing the bone marrow canal of the femur with fetal bovine serum. Cells were centrifuged, smeared on slides, dried, fixed with methanol, stained with Giemsa and analyzed at 100x (oil immersion) magnification. Analyzed were polychromatic erythrocytes (PCEs) per 1000 erythrocytes.

The positive control, 0.4 mg triethylenemelamine (TEM), induced some toxicity, as evidenced by depression of percentage of newly-formed PCEs, and TEM elevated the number of micronuclei in PCEs from male and female mice. No toxic effects were observed in the bone marrow cells and no significant number of micronuceli were induced in mice of either sex, exposed to 1581, 2811 or 5000 mg Pullulanase/kg. Pullulanase did not induce micronuceli (clastogenic effects) in male or female mice.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

October 26, 1993 - August 24, 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study was similar to OECD 475, with enzyme test material where composition is not fully described.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

The OECD guideline came later (in 1997) but the overall principles of the guideline was followed in the study.
Single treatment (IP) performed, followed by 12, 24 and 48 hour sampling of bone marrow.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: chromosome aberration assay

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

Weight: 24 to 28g
From: Charles River Laboratories, Portage, Michigan
Randomized: removed randomly from shipping cages and randomly assigned to cages
Cages: microisolator
No. of mice/cage: 5 (sex separated)
Food: Purina Rodent Laboratory Chow No. 5002C ad libitum
Water: ad libitum
Acclimatization: 6 days before dosing
Mice identification: tail mark
Room tempreature: 19-24 degreesC
Humidity: 24-68%

Route of administration:

intraperitoneal

Vehicle:

Phosphate buffered saline (PBS)

Details on exposure:

10 mL/kg
IP to 15 males and 15 females/ Pullulanase/ dose; 5 doses in total
IP to 5 males and 5 females / positive control
IP to 5 males and 5 females / vehicle control

Duration of treatment / exposure:

Pullulanase injected mice were killed 12, 24 and 48 hours after dosing (48 hours in sted of 36 hours was chosen to compare with Micronucleus test in vivo)
Positive- and vehicle control mice were killed 24 hours after dosing.
Each animal was injected with 2.5 mg colchicine/kg two hours prior to sacrifice.

Frequency of treatment:

Single injection

Post exposure period:

Mice were killed 12, 24 and 48 hours after dosing.

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

889 mg/kg bw/day (nominal)

Dose / conc.:

1 581 mg/kg bw/day (nominal)

Dose / conc.:

2 811 mg/kg bw/day (nominal)

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females/dose/time point = 15/sex/dose

Control animals:

yes

Positive control(s):

0.2 mg triethylenemelamine (TEM)/kg b.w., dosed by ip injection.
TEM and the dose 0.2 mg/kg was selected because this was known to give reproducible increase in the frequency of chromosome aberrations in Swiss Webster mice.

Tissues and cell types examined:

bone marrow from both femora

Details of tissue and slide preparation:

Both femora from mice killed by cervical dislocation were cleaned, and the bone marrow canal flushed with 1 mL disposable syringe with 25 G needle with 0.2 mL fetal bovine serum into a centrifuge tube. Cells were centrifuged for 5 min. at 100 x g, the supernatant was decanted, pellet was re-suspended in 5 mL of hypotonic solution (0.075 M KCl) for 25 minutes, at 37 degrees Celsius. Then 1 mL of fixative (3:1 methanol:acetic acid) was added, cells were centrifuged, re-suspended 2x in at least 10 mL of cold fixative, and the suspended cells were dropped onto slides.

Three slides were prepared per animal. Slides were stained for 20 min. in 5% Giemsa (in PBS with 3% methanol and 3% 0.1M citric acid), rinsed in DI water, and air dried. Slides were protected by coverslip attached with Permount, and analyzed at 100X, oil immersion, magnification.

Preliminary evaluation with uncoded slides (dose range finding):
Uncoded slides were evaluated to select the 3 highest doses per harvest time that yielded sufficient mitotic cells for analysis.

Evaluation with coded slides:
Slides were divided evenly into three sets, coded, and two same sets were scored by two cytogeneticists. The third set worked as reserve. 25 slides from the third set (5 exposed, 10 negative and 10 positive controls) were counted due to deviation of evaluation criteria.
Up to fifty cells per slide were scored by two cytogeneticists to yield 100 cells per animal, 500 cells per sex and per dose (5 mice/group).

Evaluation criteria:

For male and female mice administered with each dose, the following was calculated:
Numbers of abberation in each category (e.g. chromatid breaks, chromosome chages)
Total number of chromosome aberrations
Frequency of cells with structurallly abnormal chromosomes
The mean percent aberrant cells +/- SEM
Number of chromosome breaks /cell
Mean percent chromosome breaks/cell +/- SEM

Criteria for interpretation:
Positive response:
Dose related increase in mean % aberrant cells or mean breaks/cell observed per at least one sacrifice time, with at least one dose statistically significant.

Negative response:
When the criteria for positive response are not met.

Both biological and statistical significance were considered together.

Statistics:

Student's t-test was used to compare means of the data for the vehicle and positive control mice (significance level p<0.05).
Statistical analysis was unnecessary for Pullulanase treated groups.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative
No depression of the mitotic index and no cytogenetic damage was induced in mice of either sex, exposed to 1581, 2811 or 5000 mg Pullulanase/kg when injected by IP injection. Pullulanase did not induce chromosomal aberrations in male or female mice.

Executive summary:

Mouse bone marrow cells were examined for chromosomal aberrations. This cytogenetic study was designed to detect clastogenic effects (chromosome breaking effects).

Male and female Swiss-Webster mice (24-28g) from Charles River Laboratories, Portage, Michigan, were used in the study, after 6 day acclimatization.

In a dose range-finding study 3 male and 3 female mice/dose received vehicle (PBS 10 ml/kg), 50, 158.1, 500, 1581, or 5000 mg (half-log doses) Pullulanase/kg by IP injection. The top dose 5000mg/kg was not toxic to mice and selected as the highest dose for chromosome aberration assay.

Groups of 15 male and 15 female mice were given one of 5 doses, 500, 889, 1581, 2811 or 5000 mg Pullulanase/kg, by IP injection. The chromosome aberrations in bone marrow from animals exposed to the three highest concentrations of Pullulanase and to the vehicle and the positive control were analyzed . Five male and 5 female mice were exposed to vehicle or positive control. Bone marrow cells were collected from mice killed 12, 24 and 48 hours after exposure, by flushing the marrow canal of the femur with fetal bovine serum. Cells were centrifuged, re-suspended in 0.075 M KCl for 25 min. at 37°C, fixed with methanol:acetic acid, placed on slides, and stained with Giemsa. Bone marrow cells were evaluated for toxicity and the presence of chromosomal aberrations. Analysis was performed by 2 independent evaluators, 50 cells/slide, 100 cells/animal, 500 cells/sex/dose.

The positive control, 0.2 mg triethylenemelamine (TEM) per kg bodyweight, did not significantly depress the mitotic index of the bone marrow cells, but did induce positive responses in male and female mice. No depression of the mitotic index and no cytogenetic damage were induced in mice of either sex, exposed to 1581, 2811 or 5000 mg Pullulanase/kg. Pullulanase did not induce chromosomal aberrations in male or female mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

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