Lyase, pectate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Pectate lyase did not show mutagenic activity in the Ames assay. Pectin lyase, an enzyme from the same subclass, 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 three in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on three different enzymes, which have a genotoxicity profile comparable to pectate lyase.

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vitro, other

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 6.0, July 2017), 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, pectate lyase, has been investigated in the Ames test. A closely related enzyme, pectin lyase, was tested in the in vitro chromosome aberration test. Both tests have been performed according to current OECD guidelines, and in compliance with GLP. No evidence for genetic toxicity was observed. The results were supported by read-across from three in vitro gene mutation studies in L5178Y mouse lymphoma cells performed on three different amylases belonging to the class of glycosidases (IUBMB class 3.2.1.). The safety of the production strain is fully documented to belong to a safe strain lineage (Pariza and Johnson, 2001; Enzymes REACH Consortium, 2009) and the enzyme test material was 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 logPow 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 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) should be fully applicable. Pectate lyase is concluded not to be genotoxic. References - Pariza, M. W., and Johnson, E. A. (2001). Evaluating the Safety of Microbial Enzyme Preparations Used in Food Processing: Update for a New Century. Regulatory Toxicology and Pharmacology, 33: 173-186. - Enzymes REACH Consortium: Safety evaluation of technical enzyme products with regards to the REACH legislation. Document from Manufacturers, Importers and/or Only Representatives of one or more enzymes, who are subject to the registration requirements pursuant to REACH, 2009. http://www.enzymes-reach.org/documents.html - D. Basketter; N. Berg; F. Kruszewski; K. Sarlo; B. Concoby. The Toxicology and Immunology of Detergent Enzymes. 2012a. J. Immunotox 9(3): 320-6. - Basketter D., Berg N., Broekhuizen C., Fieldsend M., Kirkwood S., Kluin C., Mathieu S. and Rodriguez C. Enzymes in Cleaning Products: An Overview of Toxicological Properties and Risk Assessment/Management. 2012b. Reg. Toxicol. Pharmacol, 64/1: 117-123

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 January March 1999 - 19 April 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Principles of method if other than guideline:

The ‘treat and plate’ treatment method was used.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

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

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor 1254 induced Sprague Dawley rats

Test concentrations with justification for top dose:

Six doses 156, 313, 625, 1250, 2500 and 5000 μg/mL were tested.

Vehicle / solvent:

- Vehicle used: DI water
- Justification for choice of solvent/vehicle: The test substance is water-soluble and any human exposure will be in aqueous solutions.
- Solvent for positive controls: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene, N-Methyl-N-Nitro-NitrosoGuanidine

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 2 x 10^9 cells/mL

DURATION
- Preincubation period: 3 hrs (liquid culture assay).
- Exposure duration: Same as preincubation for treat and plate
- Incubation time (selective incubation) : 64 hrs

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count

Evaluation criteria:

According to the guideline.

Statistics:

N/A

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

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
- Definition of acceptable cells for analysis: Viability and gene type control

HISTORICAL CONTROL DATA
Not speficied

Conclusions:

Pectate lyase, batch PPE 6345, was not mutagenic when tested under the conditions applied in this bacterial reverse mutation test.

Executive summary:

Pectate lyase was tested in two independent experiments. The ‘treat and plate’ treatment method was used in each test to avoid the possibility that bio-available histidine in the test item might compromise the test. Six dose levels (156, 313, 625, 1250, 2500 and 5000 μg dry matter/mL) were tested. Pectate lyase was diluted in DI water was added to bacteria growth medium. The bacteria was also treated with the positive controls, respectively. After 3 hr treatment were bacteria washed, plated and incubated for 64 hours. The treatments were performed both in the absence and the presence of metabolic activation system (S-9 mix).

The test item was considered not toxic to the test bacteria, either in the absence or presence of S-9 mix. No increases over 2 -fold in the number of revertant colonies were observed in either experiment.

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

Based on the results obtained in this study, it is concluded that pectate lyase, batch PPE 6345 was not mutagenic when tested under the conditions applied in this bacterial reverse mutation test.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

17 January 2001 - 2 May 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Due to the similarities between the two enzymes, similar results are expected for pectate lyase.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Remarks:

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: human peripheral blood lymphocytes (HPBL)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Highest concentration tested was 5000 µg/mL and dilutions hereof.

Experiment 1 (3 hour exposures with 17 hour recovery in the absence and presence of S9):
-S9: Slides evaluated for 2560, 3200, 5000 ug/mL
+S9: 3200, 4000, 5000 ug/mL (repeated with 3613, 4250, 5000 ug/mL)

Experiment 2 (20 hour exposures with 0 hour recovery in the absence of S9):
-S9: Slides evaluated for 3613, 4250, 5000 ug/mL

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclohexylamine

other: 4-Nitroquinoline 1-oxyde

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 3 hours absence and presence S-9; 20 hours absence S9
- Expression time (cells in growth medium after washing): absence and presence S-9 - 17 hours, absence S9 - 0 hours. Total time for all tests was 20 hours- Selection time (if incubation with a selection agent): last 2 hours of time in medium for all tests
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

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

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: a minimum of 200 metaphase spreads containing 46 centromeres from each dose level (100 per duplicate treatment) were examined and scored for chromatid-type and chromosome-type aberrations

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

OTHER: Two hours prior to harvest, colchicine was added to the cultures at a final concentration of 0.1 μg/mL. Cells were collected by centrifugation, treated with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 3:1 v/v), capped and stored overnight or longer at 2-8°C. To prepare slides, the cells were collected by centrifugation and if necessary, the cells were resuspended in fresh fixative. Several drops of 45% (v/v) acetic acid were added to each suspension to enhance chromosome spreading. The suspension of fixed cells was applied to glass microscope slides and dried. The slides were stained for 5min in 4% (v/v) Giemsa, and mounted with coverslips.

Evaluation criteria:

A test article is considered as positive in this assay if:
1) the proportions of cells with structural aberrations at one or more concentration exceeds the normal range in both replicates, and
2) a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurs at these doses.

The assay is considered valid if the following criteria are met:
1) the binomial dispersion test demonstrates acceptable heterogeneity between replicate cultures, and
2) the proportion of cells with structural aberrations (excluding gaps) in negative control cultures falls within the normal range, and
3) at least 160 cells out of an intended 200 are analysable at each dose level, and
4) the positive control chemicals induce statistically significant increases in the number of cells with structural aberrations.

Statistics:

Fisher's exact test

Key result

Species / strain:

other: human peripheral blood lymphocytes (HPBL)

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:

TEST-SPECIFIC CONFOUNDING FACTORS: none

RANGE-FINDING/SCREENING STUDIES: experiment 1 had extended dose range to find the appropriate dose.

COMPARISON WITH HISTORICAL CONTROL DATA: yes
Appropriate negative (solvent) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges. 4-Nitroquinoline 1-oxide and cyclophosphamide were employed as positive control chemicals in the absence and presence of liver S-9 respectively. Cells receiving these were sampled in each experiment, 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations.
Treatment of cultures with Pectin Lyase in the absence and presence of S-9 (both experiments) resulted in frequencies of cells with structural aberrations that were similar to those seen in concurrent vehicle control cultures in the majority of cases. The one exception to this was observed at the highest concentration analysed (5000 mg/ml) for the continuous 20 hour treatment in the absence of S-9 in Experiment 2. However, although the number of aberrant cells (excluding gaps) exceeded the historical negative control (normal) range, the increase observed was marginal and was not present in the duplicate culture. There were no increases above normal in either replicate for cells including gaps. As such, the increase observed was considered spurious and of no biological significance.
The aberrant cell frequency for cells excluding gaps for all other Pectin Lyase treated cultures fell within normal ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1, 3+17 –S9, mitotic inhibition at highest analysed dose: 14%
Experiment 1, 3+17 +S9, mitotic inhibition at highest analysed dose: 13%
Experiment 2, 20+0 –S9, mitotic inhibition at highest analysed dose: 9%
Experiment 2, 3+17 +S9, mitotic inhibition at highest analysed dose: 0%

Conclusions:

It is concluded that pectin lyase did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to 5000 mg/ml, an acceptable top dose for chromosome aberration studies according to current regulatory guidelines, in both the absence and presence of S-9.

Executive summary:

Pectin Lyase, PPJ 6991 was tested in the chromosome aberration assay using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system. A preliminary toxicity test was performed to establish the dose range for testing in the cytogenetic test. The chromosome aberration assay was used to evaluate the clastogenic potential of the test article.

In experiment 1, the doses chosen for the 3 hour exposure in absence and presence of the metabolic activation system S-9 ranged from 274.9 to 5000 μg/mL. In experiment 2, the doses chosen for the 20 hour exposure in absence of the metabolic activation system S-9, and the 3 hour exposure in presence of the metabolic activation system S-9, ranged from 604.5 to 5000 μg/mL. All cells were harvested 20 hours after treatment initiation. Chromosome aberrations were analysed at three dose levels.

No substantial cytotoxicity was observed at any dose level. Treatment of cultures with Pectin Lyase in the absence and presence of S-9 (both experiments) resulted in frequencies of cells with structural aberrations that were similar to those seen in concurrent vehicle control cultures (p > 0.05, Fisher's Exact test). One exception was observed at the highest concentration analysed (5000 mg/ml) for the continuous 20 hour treatment in the absence of S-9 in experiment 2. The increase observed was considered spurious and of no biological significance.

Normal frequencies of cells with numerical aberrations (within historical negative control ranges), were seen following treatment with Pectin Lyase in all treated cultures.

Pectin Lyase did not induce chromosome aberrations in cultured humanperipheral blood lymphocytes when tested to 5000 mg/ml, in both the absence and presence of S-9.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across based on grouping of substances (category approach)

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

Justification for type of information:

Due to the similarities between the two enzymes, similar results are expected for pectate lyase.

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 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key 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:

Due to the similarities between the two enzymes, similar results are expected for pectate lyase.

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 6

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across based on grouping of substances (category approach)

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

Justification for type of information:

Due to the similarities between the two enzymes, similar results are expected for pectate lyase.

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

Vehicle controls validity:

valid

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Due to the lack of genetic toxicity pectate lyase is not classified.