Arabinofuranosidase, α-l-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxicity of arabinofuranosidase was tested employing two tests, Ames and Lymphocyte Micronucleus.

The Ames test resulted negative with batches PPH40331 and PPH43175.

- Arabinofuranosidase, batch PPH40331 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 (the maximum recommended concentration according to current regulatory guidelines) in the absence and in the presence of a rat liver metabolic activation system (S-9) using a modified Treat and Plate methodology.

- Arabinofuranosidase, batch PPH43175 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 (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9) using a modified Treat and Plate methodology.

Ambiguous results were seen with different batches in the micronucleus test.

- Arabinofuranosidase, batch PPH40331 did not induce biologically relevant increases in micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9). Concentrations were tested up to 5000 μg TOS/mL, a recommended regulatory maximum concentration for in vitro micronucleus assays.

- Arabinofuranosidase, batch PPH43175 showed evidence of inducing increases in micronuclei in cultured human peripheral blood lymphocytes following 24+24 hour treatment in the absence of a rat liver metabolic activation system (S-9) when tested at concentrations between 4000 to 5000 μg TOS/mL. However, these increases were poorly reproduced between three independent experimental occasions such that the biological importance was considered questionable. No such increases were observed following 3+21 hour treatments in the absence or presence of S-9 where concentrations were analysed up to 5000 μg TOS/mL (an acceptable maximum concentration for in vitro micronucleus studies according to current regulatory guidelines).

No genotoxic effects were seen with batch PPH40331 in the Ames and Lymphocyte Micronucleus assays. However, batch PPH43175 showed evidence of inducing increases in micronuclei for the two highest concentrations. To investigate this, a non-GLP study was conducted employing 3 batches, 2 samples from the batch PPH43175, batch PPH42988, and a purified batch U8DBF.

- Arabinofuranosidase, batch PPH43175 (sample 1) and arabinofuranosidase, batch PPH43175 (sample 2) showed evidence of inducing increases in micronuclei in cultured human peripheral blood lymphocytes following 24+24 hour treatment in the absence of a rat liver metabolic activation system (S-9).

- Arabinofuranosidase, batch PPH42988 showed some evidence of cytotoxicity (42% at 5000 μg TOS/mL), however, no evidence of inducing increases in micronuclei in cultured human peripheral blood lymphocytes following 24+24 hour treatment in the absence of a rat liver metabolic activation system (S-9).

- Arabinofuranosidase, batch U8DBF showed no evidence of inducing increases in micronuclei in cultured human peripheral blood lymphocytes following 24+24 hour treatment in the absence of a rat liver metabolic activation system (S-9).

To conclude, some inconsistent genotoxic potential was seen in batch PPH43175. However, when the batch was purified to only contain the active enzyme (batch U8DBF), no genotoxic effects were seen, indicating that arabinofurinosidase does not have any genotoxic potential.

The conclusion is further confirmed by read-across to mouse lymphoma test performed with enzymes closely related to arabinofuranosidase i.e three amylases and one lipase. In all tests no evidence of genetic toxicity was observed supporting the conclusion, that arabinofuranosidase is not genotoxic.

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:

14-01-2016 to 24-05-2016

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 and tryptophan locus in the genome of five strains of bacteria

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from Aroclor 1254-induced rat liver

Test concentrations with justification for top dose:

Preliminary test: No preliminary trials were carried out.
Experiment 1: 16, 50, 160, 500, 1600 and 5000 μg TOS/mL.
Experiment 2: 160, 300, 625, 1250, 2500 and 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:

4-nitroquinoline-N-oxide

2-nitrofluorene

other: N-methyl-N'-nitro- N-nitrosoguanidine, ICR-191 mutagen, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation in suspension, followed by plating on agar plates (treat and plate method)
- Cell density at seeding (if applicable): Overnight culture of approximately 10^8 - 10^9 cells/mL

DURATION
- Exposure duration, pre-incubation: The incubation mixtures were incubated with shaking at 37 ± 1 °C for 1 hour (treat and plate).
- Incubation time (selective incubation): about 72 hours

NUMBER OF REPLICATIONS: 3 for the treatments, 5 for the negative control

DETERMINATION OF CYTOTOXICITY
- Method: Following incubation, the mutation plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.

Evaluation criteria:

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

Statistics:

N/A

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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
- Water solubility: Yes
- Precipitation: Precipitation is a concentration limiting factor, but no issues were reported in this study.
- Definition of acceptable cells for analysis: Viability and gene type control

HISTORICAL CONTROL DATA
- Positive historical control data: Yes. Data fell within these ranges.
- Negative (solvent/vehicle) historical control data: Yes. Data fell within these ranges.

Conclusions:

It was concluded that arabinofuranosidase, batch PPH40331 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 (the maximum recommended concentration according to current regulatory guidelines) 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:

Arabinofuranosidase, batch PPH40331 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 arabinofuranosidase, PPH40331 is a high molecular weight protein (which may cause artefacts through growth stimulation in a standard plate-incorporation test). All arabinofuranosidase, PPH40331 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 arabinofuranosidase, PPH40331 at 16, 50, 160, 500, 1600 and 5000 μg TOS/mL, plus vehicle and positive controls. Following these treatments, no evidence of toxicity was observed in any of the tester strains.

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 160–5000 μg TOS/mL, in order to examine more closely those concentrations of arabinofuranosidase, PPH40331 approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity.

Following these treatments there was again no clear evidence of toxicity observed. 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 numbers of revertant colonies per plate all fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

Following arabinofuranosidase, PPH40331 treatments of all the test strains in the absence and presence of S-9, there were no clear and concentration-related increases in revertant numbers observed, and none 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 mutagenic activity in this assay system.

It was concluded that arabinofuranosidase, batch PPH40331 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 (the maximum recommended concentration according to current regulatory guidelines) 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 gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07-02-2017 to 07-11-2017

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 and tryptophan locus in the genome of five strains of bacteria

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from Aroclor 1254-induced rat liver

Test concentrations with justification for top dose:

Preliminary test: No preliminary trials were carried out.
Experiment 1: 16, 50, 160, 500, 1600 and 5000 μg TOS/mL.
Experiment 2: 160, 300, 625, 1250, 2500 and 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:

4-nitroquinoline-N-oxide

2-nitrofluorene

other: N-methyl-N'-nitro- N-nitrosoguanidine, ICR-191 mutagen, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation in suspension, followed by plating on agar plates (treat and plate method)
- Cell density at seeding (if applicable): Overnight culture of approximately 10^8 - 10^9 cells/mL

DURATION
- Exposure duration, pre-incubation: The incubation mixtures were incubated with shaking at 37±1°C for 1 hour (treat and plate).
- Incubation time (selective incubation): about 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Following incubation, the mutation plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.

Evaluation criteria:

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

Statistics:

N/A

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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
- Water solubility: Yes
- Precipitation: Precipitation is a concentration limiting factor, but no issues were reported in this study.
- Definition of acceptable cells for analysis: Viability and gene type control

HISTORICAL CONTROL DATA
- Positive historical control data: Yes. Data fell within these ranges.
- Negative (solvent/vehicle) historical control data: Yes. Data fell within these ranges.

Conclusions:

It was concluded that arabinofuranosidase, batch PPH43175 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 (the maximum recommended concentration according to current regulatory guidelines), 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:

Arabinofuranosidase, batch PPH43175 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 arabinofuranosidase, batch PPH43175 is an enzyme which may contain free amino acids (which may cause artefacts through growth stimulation in a standard plate-incorporation test). All arabinofuranosidase, batch PPH43175 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 arabinofuranosidase, batch PPH43175 at 16, 50, 160, 500, 1600 and 5000 μg TOS/mL. Following these treatments, no evidence of toxicity was observed.

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 160-5000 μg TOS/mL, in order to examine more closely those concentrations of arabinofuranosidase, batch PPH43175 approaching the maximum test

concentration and considered therefore most likely to provide evidence of any mutagenic activity. Following these treatments, evidence of toxicity was observed at 5000 μg TOS/mL in strain TA1535 in the absence of S-9 and TA100 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 historical ranges for vehicle control treatments, and were elevated by positive control treatments. Following arabinofuranosidase, batch PPH43175 treatments of all the test strains in the absence and presence of S-9, no notable increases in revertant numbers were observed, and none 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 mutagenic activity in this test system.

It was concluded that arabinofuranosidase, batch PPH43175 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 (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9) using a modified Treat and Plate methodology.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04-01-2016 to 29-04-2016

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

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

cultured human peripheral blood
lymphocytes

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

N/A

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The highest concentration selected for micronucleus analysis following all treatment conditions was the highest concentration tested (5000 μg TOS /mL), a recommended maximum concentration for in vitro micronucleus studies. Two lower concentrations were also analysed, 3000 and 4000 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:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: vinblastin

Details on test system and experimental conditions:

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 theculture 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+21h without S-9; 3+21h with S-9; 24+24h without S-9.
Experiment 2:3+21h with S-9.
- Expression time (cells in growth medium): 1.5-2.0 cell cycles (approximately 24 hours)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 or 48 hours

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. 4 replicas for the vehicle in the 24+24 hour treatment.

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: 2000 per concentration, 4000 for the vehicle in the 24+24 hour treatment.

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.
- 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: human lymphocytes

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 (if used)
- 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
- 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:

It was concluded that arabinofuranosidase, batch PPH40331 did not induce biologically relevant increases in micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9). Concentrations were tested up to 5000 μg TOS/mL, a recommended regulatory maximum concentration for in vitro micronucleus assays.

Executive summary:

Arabinofuranosidase, batch PPH40331 was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test

article was formulated in water for irrigation (purified water) and the highest concentration tested in the Micronucleus Experiment, 5000 μg TOS/mL (an acceptable maximum concentration for in vitro micronucleus studies according to current regulatory guidelines) was determined following a preliminary cytotoxicity Range-Finder Experiment. Treatments were conducted 48 hours after mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of arabinofuranosidase, batch PPH40331 on the replication index (RI). Micronuclei were analysed at three concentrations 3000, 4000 and 5000 μg TOS/mL. Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of

micronucleated binucleate (MNBN) cells in the vehicle cultures fell within (or very close to) the 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 study was therefore accepted as valid.

Pulse (3+21 hour) treatment of cells with arabinofuranosidase, batch PPH40331 in the absence and presence of a rat liver metabolic activation system (S-9) resulted in frequencies of MNBN cells which were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for the majority of concentrations analysed. The single exception to this was observed at the lowest concentration scored post 3+21 hour +S-9 treatment (3000 μg TOS/mL) where one of the two replicate cultures exhibited a marginal increase above the normal range. However, this increase was small with the group mean MNBN cell frequency falling within normal and no such increases observed at higher arabinofuranosidase, batch PPH40331 concentrations analysed (all replicate cultures). As such, this isolated statistical increase was not considered of biological importance.

Following extended (24+24 hour) treatment of cells in the absence if S-9, small but statistically significant increases in MNBN cell values were observed for all three replicate cultures fell within normal ranges. In addition, it was noted that the statistical significance observed post 24+24 hour –S-9 treatment was set against a low concurrent vehicle control response (0.1% MNBN cells versus historical control range of 0.1 to 0.9% MNBN cells). For these reasons the weak statistical increases observed were not considered of biological importance.

It was concluded that arabinofuranosidase, batch PPH40331 did not induce biologically relevant increases in micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9). Concentrations were tested up to 5000 μg TOS/mL, a recommended regulatory maximum concentration for in vitro micronucleus assays.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-03-2017 to 26-07-2017

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:

2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

cultured human peripheral blood
lymphocytes

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

N/A

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The highest concentration selected for micronucleus analysis following all treatment conditions was the highest concentration tested (5000 μg TOS/mL), a recommended maximum concentration for in vitro micronucleus studies.
Experiment 1: 1000, 3000, 4000 and 5000 μg TOS/mL.
Experiment 2: 3500, 4000, 4500, and 5000 μg TOS/mL.
Range-finder experiment: 3000 and 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:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: vinblastine

Details on test system and experimental conditions:

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 theculture 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+21h without S-9; 3+21h with S-9; 24+24h without S-9.
Experiment 2:3+21h with S-9.
- Expression time (cells in growth medium): 1.5-2.0 cell cycles (approximately 24 hours)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 or 48 hours

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: 2000 per concetration

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: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

positive

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: A range-finder study was conducted using 3000 and 5000 μg TOS/mL to analyse for micronuclei.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: Yes

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: The slides from the Range-Finder treatment (24+24 hour -S-9) at dose levels of 3000 and 5000 μg TOS/mL were analysed for micronuclei. A statistically significant increase in MNBN cells was observed at 5000 μg TOS/mL with a MNBN cell frequency that exceeded the normal range. No such increase was observed at the lower concentration of 3000 μg TOS/mL. However, due to the poor reproducibility of effect across three independent experimental trials using differing frozen aliquots of the provided stock of arabinofuranosidase, batch PPH43175, the biological relevance was considered questionable.
- Indication whether binucleate or mononucleate where appropriate: Yes.

HISTORICAL CONTROL DATA
- 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:

It was concluded that arabinofuranosidase, batch PPH43175 showed evidence of inducing increases in micronuclei in cultured human peripheral blood lymphocytes following 24+24 hour treatment in the absence of a rat liver metabolic activation system (S-9) when tested at concentrations between 4000 to 5000 μg TOS/mL. However, these increases were poorly reproduced between three independent experimental occasions such that the biological importance was considered questionable. No such increases were observed following 3+21 hour treatments in the absence or presence of S-9 where concentrations were analysed up to 5000 μg TOS/mL (an acceptable maximum concentration for in vitro micronucleus studies according to current regulatory guidelines).

Executive summary:

Arabinofuranosidase, batch PPH43175 was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in two experiments. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test

article was formulated in purified water and the highest concentration tested in the Micronucleus Experiments, 5000 μg TOS/mL (an acceptable maximum concentration for in vitro micronucleus studies according to current regulatory guidelines), was determined following a preliminary cytotoxicity Range-Finder Experiment. Treatments were conducted 48 hours after mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of arabinofuranosidase, batch PPH43175 on the replication index (RI). Micronuclei were analysed at three or four concentrations:

Experiment 1: 1000, 3000, 4000 and 5000 μg TOS/mL.

Experiment 2: 3500, 4000, 4500, and 5000 μg TOS/mL.

Range-finder experiment: 3000 and 5000 μg TOS/mL.

In order to further investigate an increase in micronucleated binucleate (MNBN) cells observed following the initial 24+24 hour -S-9 treatment, a second experiment was conducted (Micronucleus Experiment 2) using a separate frozen aliquot of the same batch of test article. In addition, slides from the initial Range-Finder treatment were coded and scored for MNBN cells from the 24+24 hour -S-9 treatment at 3000 and 5000 μg TOS/mL. Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in these cultures fell within the 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. All acceptance criteria were considered met and the study was therefore accepted as valid.

Experiment 1 treatments

Pulse (3+21 hour) treatment of cells with arabinofuranosidase, batch PPH43175 in the absence and presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for all concentrations analysed (both treatment regimens). The MNBN cell frequencies of all treated cultures (all concentrations) fell within the normal ranges. Extended (24+24 hour) treatment of cells with arabinofuranosidase, batch PPH43175 in the absence of S-9 (Experiment 1) resulted in frequencies of MNBN cells that were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for the two lower concentrations analysed (1000 and 3000 μg TOS/mL). At higher concentrations of 4000 and 5000 μg TOS/mL marked statistically significant increases in MNBN cell frequency was noted. Both replicate cultures at each of these concentrations exhibited MNBN cell values that clearly exceeded the normal range. A statistically significant linear trend was also apparent.

Experiment 2 treatment

A second Micronucleus Experiment (Micronucleus Experiment 2) was conducted to confirm the increases observed. This experiment consisted of a single 24+24 hour -S-9 regimen. Weak but statistically significant increases in MNBN cells were observed for concentrations of 4000 and 5000 μg TOS/mL. However, these increases were small and set against a low concurrent vehicle control response. The MNBN cell values of both replicate cultures at each of these concentrations (and all others) fell within normal values. Overall, these data alone would be considered negative.

Range-finder analysis

In order to further qualify the MNBN cell data, slides from the Range-Finder treatment (24+24 hour -S-9) at dose levels of 3000 and 5000 μg TOS/mL were analysed for micronuclei. A statistically significant increase in MNBN cells was observed at 5000 μg TOS/mL with a MNBN cell frequency that exceeded the normal range. No such increase was observed at the lower concentration of 3000 μg TOS/mL.

Overall, these data were considered to indicate evidence of a positive induction of MNBN cells following extended (24+24 hour) treatment. However, due to the poor reproducibility of effect across three independent experimental trials, the biological importance was considered questionable.

Conclusion

It was concluded that arabinofuranosidase, batch PPH43175 showed evidence of inducing increases in micronuclei in cultured human peripheral blood lymphocytes following 24+24 hour treatment in the absence of a rat liver metabolic activation system (S-9) when tested at concentrations between 4000 to 5000 μg TOS/mL. However, these increases were poorly reproduced between three independent experimental occasions such that the biological importance was considered questionable. No such increases were observed following 3+21 hour treatments in the absence or presence of S-9 where concentrations were analysed up to 5000 μg TOS/mL (an acceptable maximum concentration for in vitro micronucleus studies according to current regulatory guidelines).

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

26-06-2016 to 25-09-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A non-GLP study was performed for explorative purposes employing 3 batches, batch PPH43175 being one of them.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

GLP compliance:

no

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: cultured human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

Blood from two healthy male volunteers.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

N/A

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The test consisted of Sample 1 and Sample 2.
The highest concentration selected for micronucleus analysis following all treatment conditions was the highest concentration tested (5000 μg TOS /mL), a recommended maximum concentration for in vitro micronucleus studies. Other concentrations: 500, 1000, 2000, 3000 and 4000 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:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: vinblastin

Details on test system and experimental conditions:

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 theculture 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: 24+24h without S-9.
- Expression time (cells in growth medium): 1.5-2.0 cell cycles (approximately 24 hours)
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

SPINDLE INHIBITOR (cytogenetic assays): Not specified

STAIN (for cytogenetic assays): Not specified

NUMBER OF REPLICATIONS: A, B. Sets of duplicate cultures were exposed to the test substance. 4 replica for the vehicle.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not specified.

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

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index.
- Any supplementary information relevant to cytotoxicity: Cytotoxicity (%) is expressed as (100 – Relative Replication Index).

Evaluation criteria:

Not specified.

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:

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Not specified.

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

CYTOKINESIS BLOCK (if used)
- 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
- Positive historical control data: Yes.
- Negative (solvent/vehicle) historical control data: Yes.

Conclusions:

It is concluded that arabinofuranosidase, batch PPH43175 (sample 1) and arabinofuranosidase, batch PPH43175 (sample 2) showed evidence of inducing increases in micronuclei in cultured human peripheral blood lymphocytes following 24+24 hour treatment in the absence of a rat liver metabolic activation system (S-9).

Executive summary:

The objective of this study was to evaluate the clastogenic and aneugenic potential of arabinofuranosidase, batch PPH43175 (sample 1) and arabinofuranosidase, batch PPH43175 (sample 2), by their effects on the frequency of micronuclei in cultured human peripheral blood lymphocytes treated for 24+24 hours in the absence of a rat liver metabolising system (S-9). The concentrations tested were 500, 1000, 2000, 3000, 4000 and 5000 μg TOS /mL. Binucleate cells were analysed in cultures treated at 3000, 4000 and 5000 μg TOS/mL. The highest concentrations analysed for micronuclei induced 14% and 0% cytotoxicity, respectively.

Extended 24 hour treatment of cells with sample 1 and sample 2 in the absence of S-9 resulted in frequencies of MNBN cells which were significantly (p≤0.05) higher than those observed in concurrent vehicle controls for either all three concentrations analysed for sample 2 or for the highest two concentrations analysed for sample 1 with a concentration related effect (positive linear trend test) apparent. Although the magnitude of these increases was not large with MNBN cell frequencies for a single culture at the highest concentration analysed (5000 μg TOS/mL, sample 1), or, both replicate cultures at 4000 μg TOS/mL and a single culture at 5000 μg TOS/mL, sample 2, exceeding normal ranges. Together, these data provided evidence of a test article related effect.

It was concluded that arabinofuranosidase, batch PPH43175 (sample 1) and arabinofuranosidase, batch PPH43175 (sample 2) showed evidence of inducing increases in micronuclei in cultured human peripheral blood lymphocytes following 24+24 hour treatment in the absence of a rat liver metabolic activation system (S-9).

Reference 6

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

26-06-2016 to 25-09-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A non-GLP study was performed for explorative purposes employing 3 batches, batch PPH42988 being one of them.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

GLP compliance:

no

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: cultured human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

Blood from two healthy male volunteers.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

N/A

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The highest concentration selected for micronucleus analysis following all treatment conditions was the highest concentration tested (5000 μg TOS /mL), a recommended maximum concentration for in vitro micronucleus studies. Other concentrations: 500, 1000, 2000, 3000 and 4000 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:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: vinblastin

Details on test system and experimental conditions:

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 theculture 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: 24+24h without S-9.
- Expression time (cells in growth medium): 1.5-2.0 cell cycles (approximately 24 hours)
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

SPINDLE INHIBITOR (cytogenetic assays): Not specified

STAIN (for cytogenetic assays): Not specified

NUMBER OF REPLICATIONS: A, B. Sets of duplicate cultures were exposed to the test substance. 4 replica for the vehicle.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not specified.

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

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index.
- Any supplementary information relevant to cytotoxicity: Cytotoxicity (%) is expressed as (100 – Relative Replication Index).

Evaluation criteria:

Not specified.

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:

Metabolic activation:

with and without

Genotoxicity:

positive

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
Not specified.

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

CYTOKINESIS BLOCK (if used)
- 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
- Positive historical control data: Yes.
- Negative (solvent/vehicle) historical control data: Yes.

Conclusions:

It was concluded that arabinofuranosidase, batch PPH42988 showed some evidence of cytotoxicity (42% at 5000 μg TOS/mL), however, no evidence of inducing increases in micronuclei in cultured human peripheral blood lymphocytes following 24+24 hour treatment in the absence of a rat liver metabolic activation system (S-9).

Executive summary:

The objective of this study was to evaluate the clastogenic and aneugenic potential of arabinofuranosidase, batch PPH42988 by their effects on the frequency of micronuclei in cultured human peripheral blood lymphocytes treated for 24+24 hours in the absence of a rat liver metabolising system (S-9). Binucleate cells were analysed for micronuclei in cultures treated at 2000, 3000, 4000 and 5000 μg TOS/mL. The highest concentration, 5000 μg TOS/mL, caused 42% toxicity. Extended 24 hour treatment of cells with Arabinofuranosidase, batch PPH42988 resulted in frequencies of MNBN cells which were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for all concentrations analysed. The MNBN cell frequencies of all treated cultures (all concentrations) fell within normal ranges.

It was concluded that arabinofuranosidase, batch PPH42988 showed no evidence of inducing increases in micronuclei in cultured human peripheral blood lymphocytes following 24+24 hour treatment in the absence of a rat liver metabolic activation system (S-9).

Reference 7

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

26-06-2016 to 25-09-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A non-GLP study was performed for explorative purposes employing 3 batches, batch U8DEF being one of them.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

GLP compliance:

no

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: cultured human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

Blood from two healthy male volunteers.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

N/A

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Purified Arabinofuranosidase, batch U8DBF was evaluated over fourteen concentrations spanning a range from 17.05 to 1760 μg/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:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: vinblastin

Details on test system and experimental conditions:

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 theculture 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: 24+24h without S-9.
- Expression time (cells in growth medium): 1.5-2.0 cell cycles (approximately 24 hours)
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

SPINDLE INHIBITOR (cytogenetic assays): Not specified

STAIN (for cytogenetic assays): Not specified

NUMBER OF REPLICATIONS: A, B. Sets of duplicate cultures were exposed to the test substance. 4 replica for the vehicle.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not specified.

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

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index.
- Any supplementary information relevant to cytotoxicity: Cytotoxicity (%) is expressed as (100 – Relative Replication Index).

Evaluation criteria:

Not specified.

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:

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
Not specified.

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

CYTOKINESIS BLOCK (if used)
- 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
- Positive historical control data: Yes.
- Negative (solvent/vehicle) historical control data: Yes.

Conclusions:

It was concluded that arabinofuranosidase, batch U8DBF showed no evidence of inducing increases in micronuclei in cultured human peripheral blood lymphocytes following 24+24 hour treatment in the absence of a rat liver metabolic activation system (S-9).

Executive summary:

The objective of this study was to evaluate the clastogenic and aneugenic potential of arabinofuranosidase, batch U8DBF (a purified batch) by their effects on the frequency of micronuclei in cultured human peripheral blood lymphocytes treated for 24+24 hours in the absence of a rat liver metabolising system (S-9). Binucleate cells were analysed for micronuclei in cultures treated at 422.6, 603.7, 862.4, 1232, and 1760 μg TOS/mL. The highest concentration, 1760 μg TOS/mL, caused 1% toxicity. Extended 24 hour treatment of cells with arabinofuranosidase, purified batch U8DBF resulted in frequencies of MNBN cells which were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for all concentrations analysed. The MNBN cell frequencies of all treated cultures (all concentrations) fell within normal ranges.

It was concluded that arabinofuranosidase, batch U8DBF showed no evidence of inducing increases in micronuclei in cultured human peripheral blood lymphocytes following 24+24 hour treatment in the absence of a rat liver metabolic activation system (S-9).

Reference 8

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

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, arabinofuranosidase, has been investigated in two in vitro test systems, the Ames test, and in a cultured human peripheral blood lymphocyte micronucleus 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 four in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on three different amylases and one lipase, all belonging to the same subclass (3.1).
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 concentrate is 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 logKow 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 long 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 and lipase) should be fully applicable.
The overall conclusion is that arabinofuranosidase 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

Reason / purpose for cross-reference:

read-across source

Conclusions:

The conclusion is that the target substance arabinofuranisodase IUBMB 3.2.1.55 is not genotoxic.

Executive summary:

The present test substances, three different amylases belonging to the same subclass of glycosidases (IUBMB class 3.2.1.) as the target material arabinofuranosidase and further one lipase, belonging to the IUBMB class 3.1.1.3, 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 arabinofuranosidase IUBMB 3.2.1.55 is not genotoxic.

Reference 9

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

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:

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

See Target Record for futher information on the justification for read-across (Genetic toxicity conclusion read-across ML.00).

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 (IUBMB 3.2.1.133), 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 10

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

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 arabinofuranosidase by read-across from the in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on one amylase.

See Target Record for futher information on the justification for read-across (Genetic toxicity conclusion read-across ML.00).

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 (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 (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 11

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

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:

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

See Target Record for futher information on the justification for read-across (Genetic toxicity conclusion read-across ML.00).

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 12

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:

14 June 1989 - 11 October 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 arabinofuranosidase by read-across from the in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on one lipase.
See Target Record for futher information on the justification for read-across (Genetic toxicity conclusion read-across ML.00).

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

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: Fishers medium (10% and 20% horse serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: 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:

The highest concentration tested was 5000 µg/mL, 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.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide (without S-9) and benzo(a)pyrene (with S-9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium, growth suspension. Selection phase was performed in microtitre plates.

DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium): With exception of experiment 1 treatments in the absence of S-9 (where cultures were maintained for eight days) cultures were maintained for 7 days.
- Fixation time (start of exposure up to fixation or harvest of cells): At least 7 days after treatment.

SELECTION AGENT (mutation assays): 6-TG

NUMBER OF REPLICATIONS: duplicate

DETERMINATION OF CYTOTOXICITY
- Method: Cell density by counting viable cells, expressed as percentage relative survival (RS%)

Evaluation criteria:

A test article was considered positive if:
- The assay was valid, and
- 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
- Dose-related increases in mutation could be confirmed by regression analysis in both experiments.

Statistics:

The mutation frequency was evaluated statistically 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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No Lipase treatment in the presence of S-9 in either experiment, or in the absence of S-9 in experiment 1, resulted in a statistically significant increase in mutation frequency. Experiment 2 treatments in the absence of S-9, did result in a small but statistically significant increase in mutation frequency. However, this significant increase was observed only at the lower dose plated for determination of 6-thioguanine resistance, and furthermore, showed no dose-correlation by linear-regression analysis. The data therefore cannot be considered evidence of mutation induction at the HGPRT locus of LS178Y mouse cells.

Conclusions:

Lipase batch PPW 2771 (IUBMB 3.1.1.3) was assayed for its ability to induce mutation at the HGPRT locus (6-thioguanine resistance) in mouse lymphoma cells. It was concluded that Lipase had no mutagenic activity in the present test system.

Executive summary:

Lipase batch PPW 2771 (IUBMB 3.1.1.3) 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 (S-9 mix).

Following a wide range of treatments, separated by half-log intervals and reaching 5000 µg/mL, cells survived this dose of 5000 µg/mL (93% survival) in the absence and 500 µg/mL (11% survival) in the presence of S-9. This, together with the next three lower doses without S-9 and the next five lower doses with S-9, were plated for viability and 6-thioguanine resistance seven days after treatment (with the exception of experiment 1 treatments in the absence of S-9, plated after eight days). In the second experiment a narrower dose range was used to maximize the chance of detecting any lose related effects. The top doses plated in this experiment were 5000 µg/mL in the absence and 500 µg/mL in the presence of S-9, which yielded 104% and 5% survival, respectively.

Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S-9. Mutation frequencies in negative control cultures fell within normal ranges, 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.

No Lipase treatment in the presence of S-9 in either experiment, or in the absence of S-9 in experiment 1, resulted in a statistically significant increase in mutation frequency. Experiment 2 treatments in the absence of S-9, did result in a small but statistically significant increase in mutation frequency. However, this significant increase was observed only at the lower dose plated for determination of 6-thioguanine resistance, and furthermore, showed no dose-correlation by linear-regression analysis. The data therefore cannot be considered evidence of mutation induction at the HGPRT locus of LS178Y mouse cells.

It was concluded that Lipase had no mutagenic activity in this test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)