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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacillolysin was tested for genotoxicity employing the Ames assay.

Bacillolysin 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.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02-2010 to 07-05-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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
Target gene:
Histidine and tryptophan locus in the genome of five strains of bacteria
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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 (156, 313, 625, 1250, 2500, 5000 μg/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
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
other: ICR-191-Acridine Mutagen, N-Methyl-N'-nitro-N-nitrosoguanidine, 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 64 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.
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 if the increase is dose related and reproducible and it is not accompanied by significant increases in the viable bacterial count.
Statistics:
N/A
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
But only minor cytotoxicity was seen in the highest concentration in the absence of S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
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
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
But only minor cytotoxicity was seen in the highest concentration in the absence of S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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

Minor dose related reductions in the viable counts were observed in a few test series without the addition of the S-9 preparation. In contrast, increases in the viability were evident mainly in treatments with metabolic activation. None of those factors had significant influence on the interrelated revertant colony numbers.

Conclusions:
It was concluded that testing of bacillolysin batch PPA30428 in concentrations up to 5000 μg/mL gave no indication of mutagenic activity in the presence or absence of metabolic activation, when tested under the conditions employed in this study.
Executive summary:

Bacillolycin, batch PPA30428 was examined for mutagenic activity in the bacterial reverse mutation assay using Salmonella typhimurium strain TA1535, TA100, TA1537, TA98 and Escherichia coli WP2uvrA pKM101. Crude enzyme preparations, like the present batch of bacillolycin 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 bacillolycin 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 with 5 mg (dry matter) per ml as highest concentration. After incubation the test substance was removed by centrifugation prior to plating. 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). To confirm the result two identical and independent experiments were conducted.

A test substance is considered positive when it has induced at least a doubling in the mean number of revertants per plate compared to the appropriate solvent control in one or more of the strains, in the presence or absence of S9, if this response is dose related and reproducible. If a dose related numerical increase below a doubling but at least 50% higher than the solvent control is observed then the result is considered as equivocal and need further clarification.

No treatments of any of the Salmonella typhimurium or E. coli strains with bacillolycin, batch PPA30428 either in the presence or absence of S9 mix resulted in any increases in revertant numbers that meet these criteria for a positive equivocal response. It was concluded that testing of bacillolysin, batch PPA30428 in concentrations up to 5000 μg/mL gave no indication of mutagenic activity in the presence or absence of metabolic activation, when tested under the conditions employed in this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Not classified.