Acetylglucosaminidase, β-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Please provide version

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

Molecular formula:

Not available.

IUPAC Name:

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

Constituent 2

Reference substance name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available.

IUPAC Name:

Carbohydrates 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:

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

Molecular formula:

Not available. See remarks.

IUPAC Name:

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

Constituent 5

Reference substance name:

beta-N-acetylhexosaminidase, IUBMB 3.2.1.52

Molecular formula:

Not available

IUPAC Name:

beta-N-acetylhexosaminidase, IUBMB 3.2.1.52

Test material form:

liquid

Details on test material:

- Lot/batch No.: PPX69725
- Expiration date of the lot/batch: 25 February 2030

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:

The OECD Guideline 471 recommends a maximum test concentration for soluble non-cytotoxic substances of 5000 µg/plate (mL). The test item was tested as 5000 µg total organic solids (TOS)/mL maximum dose, equivalent to 7054 µg enzyme concentrate dry matter/mL, fulfilling the recommendation.

Test 1: 7, 21, 71, 212, 705, 2116, 7054 μg enzyme concentrate dry matter/mL
Five concentrations of the test item in the final test: (7, 212, 705, 2116, 7054 enzyme concentrate dry matter/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

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation in suspension, followed by plating on agar plates (treat and plate method)
- Cell density at seeding (if applicable): Overnight culture of approximately 1x 10^9 cells/mL

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.
If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is 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 (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes

Remarks on result:

other:

Any other information on results incl. tables

In the second test, toxicity observed as a reduction in revertant colony numbers was seen in strain TA1537 at 7054 μg enzyme concentrate dry matter/mL in the absence of S9 mix. No other signs of toxicity were observed in the second test. No precipitate was observed on any plates exposed to beta-N-acetylhexosaminidase.

Applicant's summary and conclusion

Conclusions:

It was concluded that beta-N-acetylhexosaminidase showed no evidence of mutagenic activity
in this bacterial system under the test conditions employed.

Executive summary:

In this in vitro assessment of the mutagenic potential of beta-N-acetylhexosaminidases, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to beta-N-acetylhexosaminidase diluted in water. Water was also used as a vehicle control.

The mutation tests were performed according to the treat-and-wash method. They were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. Two independent mutation tests were performed. Concentrations of beta-N-acetylhexosaminidase up to 7054 μg enzyme concentrate dry matter/mL were tested. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration. No signs of toxicity towards the tester strains were observed in the first test following exposure to beta-N-acetylhexosaminidase. In the second test, toxicity observed as a reduction in revertant colony numbers was seen in strain TA1537 at 7054 μg enzyme concentrate dry matter/mL in the absence of S9 mix. No other signs of toxicity were observed in the second test. No precipitate was observed on any plates exposed to beta-N-acetylhexosaminidase. No evidence of mutagenic activity was seen at any concentration of beta-N-acetylhexosaminidase in either mutation test. The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within the current historical control range for the laboratory.

It was concluded that beta-N-acetylhexosaminidase showed no evidence of mutagenic activity in this bacterial system under the test conditions employed