Sedolisin

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:

27 May 2015 to 26 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Revised July 1997 and issued January 1998.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of sedolisin (EC no. 848318-58-1, CAS no. 814-114-0, EC name Sedolisin, Enzyme class no 3.4.21.100 )

Molecular formula:

Not available

IUPAC Name:

Active enzyme protein of sedolisin (EC no. 848318-58-1, CAS no. 814-114-0, EC name Sedolisin, Enzyme class no 3.4.21.100 )

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.: PPF38268
- Expiration date of the lot/batch: 22-april-2025

Method

Target gene:

Histidine and tryptophan locus in the genome of five strains of bacteria

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from Aroclor 1254-induced rat liver

Test concentrations with justification for top dose:

Preliminary test: No preliminary trials were carried out.
Six concentrations of the test item (1.86, 3.73, 7.45, 14.9, 29.8, 59.6 mg enzyme concentrate dry matter/mL).
Top dose: 5000 μg/mL.

Vehicle / solvent:

Vehicle for enzyme: Deionised 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

sodium azide

other: Acrldine mutagen (ICR-191), 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation in suspension, followed by plating on agar plates (treat and plate method).

DURATION
- Exposure duration, pre-incubation: The incubation mixtures were incubated with shaking at 37 ± 1 °C for 3 hours (treat and plate).
- Incubation time (selective incubation): about 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count and observation of the bacterial background lawn. 0.1 mL aliquots of a 10^6 dilution of each bacterial suspension were poured on to minimal glucose agar plates in duplicates. The plates were inverted and incubated at 37 ± 2°C for about 72 hours and scored.

Evaluation criteria:

The test substance was considered as positive when it has induced at least a doubling in the mean number of revertant colonies per plate compared to the appertaining solvent control in one or more of the strains, in the presence or absence of S9 mix, if this response is dose related (at least 3 doses) and reproducible. In case of a dose related and reproducible numerical increase, which is below a doubling but at least 50% higher than the solvent control, the result is considered as equivocal and needs further clarification.

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:

valid

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:

valid

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:

valid

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:

valid

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:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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

HISTORICAL CONTROL DATA
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes

Applicant's summary and conclusion

Conclusions:

Based on the results obtained in this study it was concluded that Sedolisin, batch PPF38268, did not display any evidence of mutagenic activity, when tested under the conditions applied in this bacterial reverse mutation test, in the presence and absence of S9.

Executive summary:

Sedolisin, batch PPF38268 was examined for mutagenic activity in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium strain TA1535, TAl00, TA1537, TA98 and Escherichia coli WP2uvrApKM10L. Crude enzyme preparations, like the present test substance contain the free amino acids 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 the test substance in liquid culture (''treat and plate assay"). Bacteria were exposed to six doses (1.86, 3.73, 7.45, 14.9, 29.8, 59.6 mg enzyme concentrate dry matter/mL) of the test substance in a phosphate buffered nutrient broth for 3 hours. After incubation the test substance was removed by centrifugation prior to plating.

The study was conducted with and without the metabolic activation system S9 mix: a llver preparation from male rats, pre-treated with Aroclor 1254 (S9) added the cofactors required for mixed function oxidase activity. All results were confirmed by conducting two complete and independent experiments. The test substance contains an abundance of various nutrients, and composes a rich growth medium to the test bacteria. These circumstances are reflected in the present study. The test substance stimulated bacterial growth, apparent by increases of the viable cell counts. No treatments of any of the S. typhimurium and E. coli strains with the test substance resulted in any increases in the number of revertant colonies that meet the criteria for a positive or equivocal response.

Based on the results obtained in this study, it was concluded that Sedolisin, batch PPF38268 did not show any evidence of mutagenic activity when tested under the conditions applied in this bacterial reverse mutation test, in the presence and absence of S9.