Licheninase

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

 Licheninase has been investigated in two in vitro test systems, the Ames test and in a cultured human peripheral blood lymphocyte micronucleus assay. Both tests have been performed according to current OECD guidelines, and in compliance with GLP.

- It was concluded that licheninase PPB41588 did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2 uvrA pKM101) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg TOS/mL in the absence and in the presence of a rat liver metabolic activation system (S-9) using a modified Treat and Plate methodology.

- Licheninase, batch PPB41588 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). Concentrations were tested either up to 5000 µg TOS/mL (3+21 hour treatments), or to a maximum concentration limited by the presence of post-treatment precipitate (24+24 hour - S-9 treatment) in accordance with current regulatory guidelines for the in vitro micronucleus assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 July 2016 - 17 January 2017

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

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine or tryptophan locus in the genome of five strains of bacteria.

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 1254-induced rat liver, S-9 mix

Test concentrations with justification for top dose:

Experiment 1: Six concentrations of the test item (16, 50, 160, 500, 1600, and 5000 µgTOS/mL)
Experiment 2: Six concentrations of the test item (160, 300, 625, 1250, 2500 and 5000 µgTOS/mL)

Vehicle / solvent:

Vehicle for enzyme: Purified water.
Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
Solvent for the positive control: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Details on test system and experimental conditions:

The study describes experiments performed to assess the effect of licheninase in amino acid dependent strains of Salmonella typhimurium and Escherichia coli capable of detecting both induced frame-shift (TA1537 and TA98) and base-pair substitution mutations (TA1535, TA100, and WP2uvrA). The test system is a reverse mutation of amino acid dependent bacterial strains. The potential of Licheninase to induce gene mutations in the absence and presence of a rat liver metabolic activation system (S-9) using a modified Treat and Plate methodology was investigated.
DURATION
- Exposure duration, pre-incubation: 1 hour
- Incubation time (selective incubation): 3 days
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: Reduction in number of colonies and clearance of bacterial background lawn.

Evaluation criteria:

For valid data, the test article will be considered to be mutagenic in this assay if:
1. A concentration related increase in revertant numbers is ≥2-fold (in strains TA98, TA100 or WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.
2. The positive trend/effects described above are reproducible. The test article will be regarded positive in this assay if the above criteria is met. The test article will be regarded negative in this assay if the above criteria is not met.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Biological relevance will be taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments. Further experimental work may be deemed necessary to aid evaluation of the data.

Statistics:

Not performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No stated, but in the evaluation criteria it is stated that the positive controls should induce increases in revertant numbers of ≥2-fold.
- Negative (solvent/vehicle) historical control data: Yes

Conclusions:

It was concluded that licheninase PPB41588 did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2 uvrA pKM101) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg TOS/mL in the absence and in the presence of a rat liver metabolic activation system (S-9) using a modified Treat and Plate methodology.

Executive summary:

Licheninase PPB41588 was assayed for mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2 uvrA pKM101) of Escherichia coli, both in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments. A 'treat and plate' procedure was used for all treatments in this study as Licheninase PPB41588 is a high molecular weight protein (which may cause artefacts through growth stimulation in a standard plate-incorporation test).

All licheninase PPB41588 treatments in this study were performed using formulations prepared in water for irrigation (purified water). Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Licheninase PPB41588 at 16, 50, 160, 500, 1600 and 5000 μg TOS/mL, plus vehicle and positive controls. Following these treatments, evidence of toxicity was observed on the mutation plates treated at 500 μg TOS/mL and above in all the Salmonella strains when treated in the absence of S-9, and at 1600 μg TOS/mL and above in all the Salmonella strains when treated in the presence of S-9. No clear evidence of toxicity was observed following treatment of the E. coli strain at any concentration in either the absence or presence of S-9.

Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 μg TOS/mL was retained for all strains. Narrowed concentration intervals were employed covering the range 51.2-5000 μg TOS/mL, in order to examine more closely those concentrations of Licheninase PPB41588 approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. Following these treatments, evidence of toxicity was again observed only with the Salmonella strains, in this experiment at 320 μg TOS/mL and above when treated in the absence of S-9, and at 800 μg TOS/mL and above when treated in the presence of S-9. The test article was completely soluble in the aqueous assay system at all

concentrations treated, in each of the experiments performed. Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies were all comparable with acceptable ranges for vehicle control treatments, and were elevated by positive control treatments. Following Licheninase PPB41588 treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥2-fold (in strains TA98, TA100 and WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any Licheninase PPB41588 mutagenic activity in this assay system.

It was concluded that licheninase PPB41588 did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2 uvrA pKM101) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg TOS/mL in the absence and in the presence of a rat liver metabolic activation system (S-9) using a modified Treat and Plate methodology.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 16, 2016 - October 26, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2014

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

Details on mammalian cell type (if applicable):

The pooled blood of two female donors in a single experiment.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Highest concentration tested was 5000 µg TOS/mL (stock solution 50 mg/mL weighed out as received) and dilutions hereof: 18.14, 30.23, 50.39, 83.98, 140, 233.3, 388.8, 648, 1080, 1800, 3000, 5000 µg TOS/mL.

Vehicle / solvent:

Vehicle for enzyme: Purified water. Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
Solvent for the positive control: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Vinblastine

Details on test system and experimental conditions:

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.

METHOD OF APPLICATION: Cells were placed in HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin. The mitogen Phytohaemagglutinin 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.

DURATION
- Preincubation period: Blood cultures were incubated at 37±1°C for approximately 48 hours and rocked continuously.
- Exposure duration: Experiment 1: 3+21, -S-9; 3+21, +S-9; 24+24, -S-9. Experiment 2:3+21, +S-9
- Fixation time (start of exposure up to fixation or harvest of cells): Centrifuged for 10 min, cells swelling by KCl for 4 min, centrifuging again for 10 min and an additional 2-3 minutes if necessary, until the cell pellets were clean.

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): Acridine Orange

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

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Lymphocytes were kept in fixative at 2-8°C prior to slide preparation for a minimum of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged and resuspended in a minimal amount of fresh fixative. Cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were stained by immersion in 125 μg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 seconds, washed with PBS (with agitation) for a few seconds before transfer and immersion in a second container of PBS for approximately 10 minutes. Slides were air-dried and stored protected from light at room temperature prior to analysis.

NUMBER OF CELLS EVALUATED: 1000 per concetration, 2000 for the vehicle

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Binucleate cells were only included in the analysis if all of the following criteria were met:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.
A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index. S-9 mix or KCl (0.5 mL per culture) was added appropriately. Cultures were treated with the test article, vehicle (1 mL per culture). Positive control treatments were not included. The final culture volume was 10 mL. Cultures were incubated at 37±1°C for the designated exposure time. 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.
- Any supplementary information relevant to cytotoxicity: Cytotoxicity (%) is expressed as (100 – Relative Replication Index).

Evaluation criteria:

For valid data, the test article was 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 MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells 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.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result

Statistics:

The proportion of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test. Probability values of p≤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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No marked changes in osmolality (shifts of greater than 50 mOsm/kg) or pH (shifts of greater than 1 pH unit) were observed at the highest concentration tested (5000 μg/mL) as compared to the concurrent vehicle controls
- Evaporation from medium: N/A
- Water solubility: Enzymes are water soluble.
- Precipitation: Enzymes are water soluble.
- Definition of acceptable cells for analysis: The cytoplasm remained essentially intact, and the daughter nuclei were of approximately equal size.

RANGE-FINDING/SCREENING STUDIES: Cytotoxicity range finding was conducted.

CYTOKINESIS BLOCK
- Distribution of mono-, bi- and multi-nucleated cells: Yes

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: Not statistically significant
- Indication whether binucleate or mononucleate where appropriate: Yes.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: The proportion of micronucleated binucleate cells in the vehicle cultures fell within current 95th percentile of the observed historical vehicle control (normal) ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Slides from the cytotoxicity Range-Finder Experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) was determined. Cytotoxicity (%) is expressed as (100 – Relative RI). A selection of random fields was observed from enough treatments to determine whether chemically induced cell cycle delay or cytotoxicity had occurred.

Conclusions:

Licheninase, batch PPB41588 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). Concentrations were tested either up to 5000 µg TOS/mL (3+21 hour treatments), or to a maximum concentration limited by the presence of post-treatment precipitate (24+24 hour - S-9 treatment) in accordance with current regulatory guidelines for the in vitro micronucleus assay.

Executive summary:

The clastogenic and aneugenic activity of licheninase, batch PPB41588 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.

Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in the cultures fell within current 95th percentile of the observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment. All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei. The proportion of binucleate cells with micronuclei in all cultures of the vehicle controls (purified water) was within the limits of the historical ranges.

Following 3+ 21 hour - S-9 treatment small but statistically significant increases were observed at the intermediate and high concentrations analysed. However, these increases were small such that all licheninase, batch PPB41588 treated cultures for all concentrations analysed (all treatments) exhibited MNBN cell frequencies that fell within normal ranges. The statistical increases observed were also noted to be set against a low concurrent vehicle control response. As such, these small statistical increases were not considered of biological importance.

The 24 + 24 hour treatment of the cells resulted in MNBN cells that were generally similar and not significantly higher than those observed in concurrent vehicle controls.

It is concluded that licheninase, batch PPB41588 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). Concentrations were tested either up to 5000 μgTOS/mL (3+21 hour treatments), or to a maximum concentration limited by the presence of post-treatment precipitate (24+24 hour – S-9 treatment) in accordance with current regulatory guidelines for the in vitro micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

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