Licheninase

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 July 2016 - 17 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Licheninase

EC Number:

819-930-0

Cas Number:

37288-51-0

Molecular formula:

Not applicable, please see remarks

IUPAC Name:

Licheninase

Constituent 2

Reference substance name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available

IUPAC Name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 3

Reference substance name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 4

Reference substance name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Constituent 5

Reference substance name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Test material form:

liquid

Details on test material:

- Lot/batch No.: PPB41588
- Expiration date of the lot/batch: 05-07-2026
- Storage condition of test material: < -10⁰C

Method

Target gene:

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

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver, S-9 mix

Test concentrations with justification for top dose:

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

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Details on test system and experimental conditions:

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

Evaluation criteria:

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

Statistics:

Not performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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

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

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

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

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

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

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

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