Laccase

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No mutagenic activity of laccase could be detected in the Ames Assay. Laccase did not induce chromosomal aberrations in the in vitro mammalian chromosome aberration test performed with human lymphocytes. These results are supported by read-across from several in vitro gene mutation studies in L5178Y mouse lymphoma cells performed with different enzymes. One of these read-across studies are performed with an enzyme which is belonging to the same enzyme IUB-class, the oxidoreductases (EC 1), as laccase.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 May 1998 - 27 August 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Principles of method if other than guideline:

Preliminary investigations demonstrated that laccase significantly increased the growth of the histidine requiring Salmonella strains following direct plate incorporation. To overcome this problem all Salmonella strains were exposed to laccase in liquid culture (“treat and plate assay”). Bacteria were exposed to 6 doses of the test substance in the nutrient broth for 3 hours with 5 mg test material per ml as highest concentration. After incubation the test substance was removed by centrifugation prior to plating.
The part of the study comprising Escherichia coli was conducted, using the direct plate incorporation assay. Six doses of the test substance were applied with 5 mg test material per plate as the highest dose level.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The study describes experiments performed to assess the effect of the test material laccase in amino acid dependent strains of Salmonella typhimurium and Escherichia coli capable of detecting both induced frame-shift (TA1537 and TA98) and base-pair substitution mutations (TA1535, TA100, and WP2uvrA). The test system is a reverse mutation of amino acid dependent bacterial strains.

Species / strain / cell type:

bacteria, other: Salmonella typhimurium TA1537, TA98, TA1535, TA100, Escherichia coli WP2uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor 1254 induced SPF Sprague Dawley rats obtained from ICN Pharmaceuticals and the co-factors required for mixed function oxidase activity (S9 mix).

Test concentrations with justification for top dose:

The top dose applied was 5 mg test substance per ml for the Salmonella strains and 5 mg per plate for the E. coli strain (5 mg test substance is equivalent to 0.83 mg total organic solids, TOS). Further doses were 2-fold dilutions hereof, with and without the metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile deionised water
- Justification for choice of solvent/vehicle: The test 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: N-Methyl- N’-Nitro-Nitrosoguanidine, N-Ethyl- N’-Nitro-Nitrosoguanidine, 2-nitrofluorene, 9-aminoacridine, 2-aminoanthracene, Benzo(alpha)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: For the Samonella strains, all applications were done in medium before plating, i.e a liquid culture assay (treat and plate assay), while the direct plate incorporation procedure was used for the E. coli strain.

DURATION
- Exposure duration: 3 hrs (liquid culture assay, Salmonella strains); 64 hrs ( direct plate incorporation, E. coli)
- Incubation time (selective incubation) : 64 hrs

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count

Evaluation criteria:

The numbers of revertant colonies at each treatment test point were compared to the corresponding negative control values for each set of triplicate plates.
The tests were considered to be valid as all the following criteria were met:
- negative and positive control data were consistent with the historical control data for this laboratory
- the positive control data showed marked increases over the concurrent negative control values
- the evaluation of the data was not restricted by loss of plates (e.g. through contamination).
The test item would have been considered to have shown evidence of mutagenic activity in this study if all of the following criteria had been met:
- increases in the numbers of revertant colonies were observed at one or more test points
- the mean number of revertant colonies at the test point showing the largest increase was more than twice the corresponding negative control value for the Salmonella strains TA 1535, TA 1537 and TA 98, or showing a 50% increase over control value for TA 100 and E. coli WP2uvrA
- the effect of the test item was dose related and reproducible

Statistics:

Statistical evaluation was not judged to provide any added value and was therefore not performed.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The test item was not toxic to the test bacteria: no marked reductions in the number of revertant colonies or growth of the background lawn of non-revertant bacteria were observed, compared to the negative control plates.
On the basis of these results, 5 mg test substance/mL or plate was chosen as the highest dose level for the main tests.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and positive control values were compatible with the historical control values for this laboratory.

Conclusions:

No dose-related and reproducible increases in revertants were obtained with any of the bacterial strains exposed to laccase, either in the presence or absence of S-9 mix.
It was concluded, that the results of the experiments gave no indication of mutagenic activity of laccase in the presence or absence of metabolic activation.

Executive summary:

Laccase, batch PPX5720, was examined for mutagenic activity in the bacterial reverse mutation assay using Salmonella typhimurium strain TA1535, TA100, TA1537 and TA98 and Escherichia coli WP2uvrA.

Crude enzyme preparations, like the present batch may contain the free amino acid histidine and/or tryptophan , which exceeds the critical concentration for incorporation in the direct standard assay. Preliminary investigations demonstrated that laccase significantly increased the growth of the histidine requiring Salmonella strains following direct plate incorporation while the part of the study comprising Escherichia coli could be conducted, using the direct plate incorporation assay. To overcome the problem with the content of histidine, all Salmonella strains were exposed to laccase in liquid culture (“treat and plate assay”). Bacteria were exposed to 6 doses of the test substance in the nutrient broth for 3 hours with 5 mg test material per ml as highest concentration. After incubation the test substance was removed by centrifugation prior to plating. The direct plate incorporation assay included 6 doses of the test substance with 5 mg test material per plate as the highest dose level.

The study was conducted with and without the metabolic activation system S9 - a liver preparation from male rats, pre-treated with Aroclor 1254, and the co-factors required for mixed function oxidase activity (S9 mix).

Two identical and independent experiments were conducted.

The treatment of the Salmonella and E.coli strains with laccase in the presence or absence of S9 mix, did not result in any increases in revertant numbers.

In conclusion, laccase was found not mutagenic.

 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 November 1996 - 17 July 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate included in the study report.

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Based on a preliminary toxicity test, the following range of concentrations of Laccase, batch PPX 5720, was used for the 2 main tests :
without S-9 mix: between 12.5 and 400 ug TOS*/mL
with S-9 mix : between 250 and 5000 ug TOS/mL
*TOS = Total Organic Solids;

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Cell culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: without S-9 mix: Mitomycin C; with S-9 mix: cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in suspension

DURATION
- Exposure duration: Cultures, established approx. 48 h before testing, were treated for 3 h in the presence of S9-mix or continuously for 19 or 43 hrs in the absence of S-9 mix. All cultures were harvested after 19 and 43 hours.

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

NUMBER OF REPLICATIONS: Duplicate cultures.

NUMBER OF CELLS EVALUATED: at least 1000 cells scored per culture; chromosomal aberrations scored based on 100 metaphases per culture (wherever possible)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

It would have been concluded that the test item had shown clastogenic activity in this study if all of the following criteria had been met:
- the increases in the frequency of metaphases with aberrant chromosomes were observed at one or more test concentrations
- the increases were reproducible between replicate cultures and between tests and must exceed the historical range at this laboratory
- the increases were statistically significant
- the increases were not associated with large changes in osmolarity of the treated cultures
The historical negative control data for this laboratory was also considered in the evaluation.
The test item would have been considered to have given a negative response if no reproducible, statistically significant increases were observed.
Results which failed to meet the stated criteria for a negative or positive response would have been considered to be equivocal.

Statistics:

The statistical analysis was performed using Fischer’s Exact Probability Test when the observed events all fell into one or other of two mutually exclusive classes.
The frequency of aberrant metaphases for each treatment group was compared with the corresponding solvent group value using a one-tailed test.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA:
Small, but statistically significant increases in the frequency of metaphases with chromosomal aberrations were observed at several test points, compared to the solvent control values. These increases were not considered to be biologically significant for the following reasons :
When gap-type aberrations were included in the analysis, four cultures had frequencies of aberrant metaphases which narrowly exceeded the historical control range at this laboratory (0-9%). However, gaps were of questionable significance.
When gaps were excluded, only one culture had a frequency value (5%) which exceeded the historical control range (0-4%).
The increases, excluding gaps, were poorly reproducible between the two main tests.
The results fit the stated criteria for a negative response.

The negative controls were within the historical negative control ranges.
Normal frequencies of cells with numerical aberrations were seen under all treatment conditions.
The positive controls induced satisfactory levels of aberrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY: At the highest concentrations without S-9 mix, laccase produced appropriate reductions in mean mitotic index (42-69%) compared to controls. When treated with S-9 mix, only top dose cultures (5 mg/mL) showed reductions in the mitotic index.

Conclusions:

It is concluded that laccase did not cause chromosomal aberrations in this in vitro cytogenetic test using cultured human lymphocytes.

Executive summary:

The clastogenic potential of the test substance laccase was evaluated by its effect on chromosomes of human peripheral blood lymphocytes according to OECD guideline 473 (1983).

The test item was tested in human lymphocytes in primary cultures of whole blood in the absence and presence of S-9 mix. The cultures were treated with formulations of the test item in cell culture medium. A preliminary and 2 main, independent tests were performed.

Cultures, established approx. 48 h before testing, were treated for 3 h, in the presence of S-9 mix, or continuously for 19 or 43 hrs in the absence of S-9 mix. Cultures were harvested after 19 and 43 hours.

Based on a preliminary toxicity test, the following range of concentrations of Laccase, batch PPX 5720, was used for the 2 main tests:

without S-9 mix: between 12.5 and 400 ug TOS (Total Organic Solids)/mL

with S-9 mix: between 250 and 5000 ug TOS/mL

 

Small, but statistically significant increases in the frequency of metaphases with chromosomal aberrations were observed at several test points, compared to the solvent control values. These increases were not considered to be biologically significant for the following reasons :

When gap-type aberrations were included in the analysis, four cultures had frequencies of aberrant metaphases which narrowly exceeded the historical control range at this laboratory (0-9%). However, gaps were of questionable significance.

When gaps were excluded, only one culture had a frequency value (5%) which exceeded the historical control range (0-4%).

The increases, excluding gaps, were poorly reproducible between the two main tests.

The results fit the stated criteria for a negative response.

The positive controls induced satisfactory levels of aberrations.

 

It was concluded that laccase under the conditions of test, did not show any evidence of clastogenic activity.

evidence of clastogenic activity.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb. 17, 1992 - Aug. 25, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was a GLP study conducted in compliance with OECD guideline 476.

Qualifier:

according to guideline

Guideline:

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

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

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 days
- Expression time (cells in growth medium): 2 days
- 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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The Glucose Oxidase, PPX 3679, 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 heat-inactivated glucose oxidase, PPX 3679 was assayed for 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).

 

It was decided to heat-inactivate the test compound for the following reasons:

1. Glucose oxidase catalyses the formation of hydrogen peroxide from D-glucose.

2. It is well known from the literature that hydrogen peroxide is a very potent cytotoxic compound and the formation of free radicals like the hydroxyl radical is responsible for the induction of mutation of the base-pair substitution type.

3. Lastly, in an initial range-finder experiment, it was observed that the metabolic activation system (S-9) was partly inactivated by the test substance.

 

Following a wide range of treatments in the range-finder experiment, 4 doses were chosen for the first experiment, ranging from 2000 to 5000 µg/mL. All doses were plated for viability and 5-trifluorothymidine resistance 2 days after treatment. The top dose yielded 105.1% and 103.6% relative survival in the absence and presence of S-9.

 

In the second experiment the same dose range was selected. The top dose 5000 µg/mL yielded 72.1% and 93.8% relative survival with and without S-9, respectively.

 

In the absence of S-9, no significant increases in mutant frequency were obtained in experiment 1 following treatment with the test substance at any dose level. Over the whole dose range, however, a weak dose-related effect was indicated by the test for linear trend. In experiment 2 no significant increase in mutant frequency were observed and there was no indication of a dose-response.Therefore it is considered that the weak dose-response observed in experiment 1occurred by chance and little biological significancecan be attached to it.

 

In the presence of S-9, a statistically significant increase in mutant frequency was observed at the top dose of 5000 µg/mL in experiment 1 and a dose-response was indicated. In experiment 2 the same dose range was employed and no statistically significant increases were observed and there was no indication of a dose-response. Therefore the experiment 1 findings were not reproducible and are attributed to a chance effect of no biological significance.

 

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.

 

It is concluded that, under the conditions employed in this study, the heat-activated test substance had no mutagenic activity when tested up to 5000 μg/mL in either the absence or presence of S-9

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jun. 14, 1989 - Oct. 10, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT (6-thioguanine resistance)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

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

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

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

Details on test system and experimental conditions:

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

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

SELECTION AGENT: 6-TG

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

Evaluation criteria:

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

Statistics:

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

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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

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

Conclusions:

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

Executive summary:

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

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

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

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

Reference 5

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:

Oct. 25, 1993 - Sept. 14, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was a GLP study conducted in compliance with OECD guideline 476.

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.

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 6

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:

Jan. 11, 1990 - Aug. 20, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT (6-thioguanine resistance)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

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

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

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

Details on test system and experimental conditions:

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

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

SELECTION AGENT : 6-TG

NUMBER OF REPLICATIONS: Preliminary trial and two independant replicates.

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

Evaluation criteria:

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

Statistics:

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

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

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

Executive summary:

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

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

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

 

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

 

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

 

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

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From January 29, 1996 to March 18, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to GLP and the procedures were according to the OECD test guideline 471 (1983).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Annex to Commission Directive 92/69/EEC of 31 July 1992. B.14. L 383 A/160 and OECD Guidelines for the Testing of Chemicals, 471, 1983 with the following adaptation:
Crude enzyme preparations, like the present batch of Laccase, contain the free amino acid histidine and tryptophan, most often in an amount which exceeds the critical concentration for incorporation in the direct standard assay.
To overcome this problem, all strains were exposed to Laccase in liquid culture (“treat and plate assay”).

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

The study describes experiments performed to assess the effect of laccase in amino acid dependent strains of Salmonella typhimurium capable of detecting both induced frame-shift (TA1537 and TA98) and base-pair substitution mutations (TA1535 and TA100). The test system is a reverse mutation of amino acid dependent bacterial strains.

Species / strain / cell type:

bacteria, other: Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S-9 mix

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 20.6 , 41.3 ,82.5, 165, 330, 660 µg TOS (total organic solids)/mL
Concentration range in the main test (without metabolic activation): 5.2, 10.3, 20.6 , 41.3 ,82.5, 165, 330, 660 µgTOS (total organic solids)/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile deionized 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: without S-9 mix: N-Methyl- N’-Nitro-Nitrosoguanidine , 2-nitrofluorene, 9-aminoacridine; with S-9 mix: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium before plating, i.e a liquid culture assay (treat and plate assay).

DURATION
- Exposure duration: 3 hours
- Incubation time (selective incubation): 64 hrs

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count

Evaluation criteria:

The numbers of revertant colonies at each treatment test point were compared to the corresponding negative control values for each set of triplicate plates.
The tests were considered to be valid as all the following criteria were met:
- negative and positive control data were consistent with the historical control data for this laboratory
- the positive control data showed marked increases over the concurrent negative control values
- the evaluation of the data was not restricted by loss of plates (e.g. through contamination).
The test item would have been considered to have shown evidence of mutagenic activity in this study if all of the following criteria had been met:
- increases in the numbers of revertant colonies were observed at one or more test points
- the mean number of revertant colonies at the test point showing the largest increase was more than twice the corresponding negative control value
- the effect of the test item was dose related and reproducible

Statistics:

Statistical evaluation was not judged to provide any added value and was therefore not performed.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 660 µg TOS (total organic solids)/mL)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The number of revertant colonies on the control plates were within the normal range of the historical data. The test item had no significant toxic effect on the strains, however, decreases in the viable count were observed at the highest dose levels in tests with TA1535 and TA100 without S9, but only significant with TA100 at doses higher than 2.5 mg per mL.
A standard solution of 10% w/v of the test material was prepared. This was further diluted 10% in the liquid culture phase, providing a maximum concentration 10 mg/mL (equivalent to 0.66 mg total organic solids (TOS) per mL) during the exposure of the test system.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and positive control values were compatible with the historical control values for this laboratory when liquid culture assay is applied.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Based on the results obtained in this study, it is concluded that the test substance laccase is not mutagenic in the Ames test.

Executive summary:

Laccase was examined for mutagenic activity in the bacterial reverse mutation assay using Salmonella typhimurium strain TA1535, TA100, TA1537 and TA98.

Crude enzyme preparations, like the present batch contain the free amino acid histidine and tryptophan, which exceeds the critical concentration for incorporation in the direct standard assay. To overcome this problem all strains were exposed to the test substance in liquid culture (“treat and plate assay”).

Bacteria were exposed to 6 doses of the test substance in a phosphate buffered nutrient broth for 3 hours with10 mg test substance/mL, equivalent to 0.66 mg TOS/mL as highest concentration. After incubation the test substance was removed by centrifugation prior to plating. The number of revertants to prototrophy and viable cells were estimated.

The study was conducted in the presence and absence of metabolic activation - a liver preparation from male rats, pre-treated with Aroclor 1254, and the cofactors required for mixed function oxidase activity (S-9 mix).

All results were confirmed by conducting two complete and independent experiments.

The sensitivity of the individual bacterial strains was confirmed by significant increases in number of revertant colonies induced under similar conditions by diagnostic mutagens.

 

No dose-related and reproducible increases in revertants to prototrophy were obtained with any of the bacterial strains exposed to laccase, either in the presence or absence of S-9 mix.

 

It was concluded, that the results of the experiments gave no indication of mutagenic activity of laccase in the presence or absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:
The genetic toxicity of laccase has been investigated in two test systems, the Ames test (two studies) and the in vitro chromosome aberration test. 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 several in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on different enzymes. One of these read-across studies are performed with an enzyme which is belonging to the same enzyme IUB-class, the oxidoreductases (EC 1), as laccase.

 

Moreover, the safety of the production strain is fully documented to belong to a safe strain lineage (Pariza and Johnson, 2001) and the enzyme concentrate is well characterized. Because enzymes are built up of the same amino acids the physical and chemical characteristics will be very similar for different enzymes, and hence read-across from other non-proteolytic enzymes (e.g. amylase) should be fully applicable.

 

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

 

Reference:

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

Justification for selection of genetic toxicity endpoint
No genetic toxicity of laccase could be detected in an in vitro gene mutation study in bacteria (two studies) or in vitro cytogenicity study in mammalian cells. The production strains of laccase further meet the criteria for a safe strain production micro-organism. The conclusion that laccase is non-genotoxic is supported by the read-across from in vitro gene mutation studies in mouse lymphoma cells of other enzymes (including one oxidoreductase).

Justification for classification or non-classification

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