Alpha,alpha-trehalaseIUBMB 3.2.1.28

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Trehalase was tested for genotoxicity in vitro using Ames and micronuclus assay.

- It was concluded that trehalase, batch PPT48569 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.

- It is concluded that trehalase, batch PPT42347 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.

- The conclusion is further confirmed by read-across to mouse lymphoma test performed with enzymes closely related to trehalase i.e three amylases and one lipase. In all tests no evidence of genetic toxicity was observed supporting the conclusion, that trehalase 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:

09-11-2017 to 18-01-2018

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, 5000 μg/mL
Experiment 2: 160, 300, 625, 1250, 2500, 5000 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:

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 to 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).
- Exposure duration: Same as preincubation for treat and plate
- Expression time (cells in growth medium): 3 days
NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: The background lawns of the plates were examined for signs of toxicity and the revertant counts assessed for any marked reduction in revertants compared to the concurrent vehicle controls.

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 trends/effects described above were reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were 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:

Not performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

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 (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes

Conclusions:

It was concluded that trehalase, batch PPT48569 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:

Trehalase, batch PPT48569 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 trehalase, batch PPT48569 may contain free amino acids i.e. histidine and tryptophan (which may cause artefacts through growth stimulation in a standard plate-incorporation test).

All trehalase, batch PPT48569 treatments in this study were performed using formulations prepared in water for irrigation (purified water).

Mutation Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of trehalase, batch PPT48569 at 16, 50, 160, 500, 1600 and 5000 μg TOS/mL. Following these treatments, evidence of toxicity was observed in strain TA98 in the absence of S-9 only at 5000 μg TOS/mL.

Mutation 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 trehalase, batch PPT48569 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 strains TA100 and TA1535 in the absence of S-9, and in strain TA98 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 each experiment. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments. Following trehalase, batch PPT48569 treatments of all the test strains in the absence and presence of S-9, no notable and concentration-related 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 trehalase, batch PPT48569 mutagenic activity in this assay system.

It was concluded that trehalase, batch PPT48569 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 cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-12-2016 to 16-03-2017

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: cultured human peripheral bloodlymphocytes

Details on mammalian cell type (if applicable):

Blood from two healthy, non-smoking female 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 (OECD, 2010). Other concentrations included 1000, 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: 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:
Range-finders experiment: 3+21h without S-9; 3+21h with S-9; 24+24h without S-9.
Experiment: 3+21h without S-9; 3+21h with S-9; 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): 24 or 48 hours

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): Acridine Orange

NUMBER OF REPLICATIONS: 2 replicates for treatments and 4 for controls.

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 (approximately 1250 g, two to three minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were air-dried then stored protected from light at room temperature prior to staining. Slides were stained by immersion in 12.5 μg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 minutes and washed with PBS (with agitation) for a few seconds. The quality of the stain was checked. Slides were air-dried and stored protected from light at room temperature. Immediately prior to analysis 1-2 drops of PBS were added to the slides before mounting with glass coverslips.

NUMBER OF CELLS EVALUATED: 1000 per concetration and 2000 for the controls.

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. Cochran-Armitage trend test was applied to each treatment condition. Probability values of p≤0.05 were accepted as significant.

Key result

Species / strain:

lymphocytes: cultured human peripheral bloodlymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Small but not biologically significant increases for 3+21 hour +S-9 treatment (3000 and 4000 μg TOS/mL) and 24+24 hour -S-9 treatment (5000 μg TOS/mL).

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

22% cytotoxicity was seen at 24+24 Hour Treatments -S-9, although the values fell within historical ranges.

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. Not a controbuting factor in this study.
- Definition of acceptable cells for analysis: The cytoplasm remained essentially intact, and the daughter nuclei were of approximately equal size.

RANGE-FINDING/SCREENING STUDIES: Yes

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. Binucleate.

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.

Treatment of cells with trehalase, batch PPT42347 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 the majority of concentrations analysed. Exceptions to this were observed for the lowest two concentrations analysed post 3+21 hour +S-9 treatment (3000 and 4000 μg TOS/mL) and the highest concentration analysed post 24+24 hour -S-9 treatment (5000 μg TOS/mL) where statistically significant increases were observed. However, in all instances, these statistical increases were small with both replicate cultures at each concentration exhibiting MNBN cell values that fell within the 95th percentile of the normal ranges. The MNBN cell values of all other treated cultures (all concentrations, all treatment conditions) also fell within normal values. As such, these statistical increases were considered of no biological importance.

Conclusions:

It was concluded that trehalase, batch PPT42347 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:

Trehalase, batch PPT42347 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 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 trehalase, batch PPT42347 on the replication index (RI). Micronuclei were analysed at 1000, 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 cultures fell within current 95th percentile of the observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment. All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei. All acceptance criteria were considered met and the study was therefore accepted as valid.

Treatment of cells with trehalase, batch PPT42347 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 the majority of concentrations analysed. Exceptions to this were observed for the lowest two concentrations analysed post 3+21 hour +S-9 treatment (3000 and 4000 μg TOS/mL) and the highest concentration analysed post 24+24 hour -S-9 treatment (5000 μg TOS/mL) where statistically significant increases were observed. However, in all instances, these statistical increases were small with both replicate cultures at each concentration exhibiting MNBN cell values that fell within the 95th percentile of the normal ranges. The MNBN cell values of all other treated cultures (all concentrations, all treatment conditions) also fell within normal values. As such, these statistical increases were considered of no biological importance.

It was concluded that trehalase, batch PPT42347 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 3

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, Trehalase, 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.
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 trehalase 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

Conclusions:

The conclusion is that the target substance trehalase IUBMB 3.2.1.28 is not genotoxic.

Executive summary:

The present test substances, three different amylases and further one lipase 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 trehalase IUBMB 3.2.1.28 is not genotoxic.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

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 trehalase 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 5

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 trehalase 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 6

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 trehalase 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 7

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 trehalase 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)

Justification for classification or non-classification

Not classified.