Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 July 2012 - 8 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July, 1997
Deviations:
no
Principles of method if other than guideline:
The ‘treat and plate’ method was used in each test to avoid the possibility that bio-available histidine in the test item might compromise the test.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Brownish liquid at room temperature
- Lot/batch No.: PPY32896
- Expiration date of the lot/batch: 07 Feb 2022
- Stability under test conditions: The test material and dilution (10%) were stable for at least 24 hours at 5 degrees Celcius.
- Storage condition of test material: minus 20 degrees of Celcius.

Method

Target gene:
Histidine and 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:
S9 from Aroclor 1254
Test concentrations with justification for top dose:
Six doses 156, 313, 625, 1250, 2500 and 5000 μg TOS/mL were tested. Highest dose enzyme activity.
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
Positive controls:
yes
Positive control substance:
2-nitrofluorene
other: Acridine mutagen (ICR-191), 1-Methyl-3-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 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: 0.1 mL aliquots of a 10^6 dilution of each bacterial suspension were poured on to minimal glucose agar plates (added the required amino acids in excess) in duplicates.
Evaluation criteria:
A test substance is considered positive when it has induced at least a doubling in the mean number of revertant colonies per plate compared to the appropriate 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:
N/A

Results and discussion

Test results
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
Remarks:
Some cytoxocity was seen in the absence of S9 for TA100 in the highest concentration and TA1537 in the highest 3 concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Some cytotoxicity was seen in the absence of S9 for TA100 in the highest concentration and TA1537 in the highest 3 concentrations.
Growth stimulation (feeding effect) was present in a few test series especially in with the E. coli strain with addition of S9 as demonstrated by increases in the viable count of exposed cultures compared to the solvent control. As a consequence, weak increases in the number of spontaneous revertant colonies were present which obviously is without biological significance.

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:
The results of the bacterial mutagenicity tests gave no indication of the presence of mutagenic components in amylase, batch PPY32896, when tested under the conditions applied in this study, in the presence and absence of S-9.
Executive summary:

Amylase, batch PPY32896 was examined for mutagenic activity in the bacterial reverse mutation assay using Salmonella typhimurium strains TA1535, TA100, TA1537, TA98 and Escherichia coli WP2uvrApKM101. Crude enzyme preparations, like the present batch of amylase 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 amylase 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 5000 μg TOS/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). All results were confirmed by conducting two complete and independent experiments.

Amylase contains an abundance of various nutrients, and composes a rich growth medium to the test bacteria. These circumstances are reflected in the present study. No toxicity of the test substance to the bacteria was observed.

Growth stimulation (feeding effect) was present in a few test series especially in with the E. coli strain with addition of S9 as demonstrated by increases in the viable count of exposed cultures compared to the solvent control. As a consequence weak increases in the number of spontaneous revertant colonies were present which obviously is without biological significance.

The results of the bacterial mutagenicity tests gave no indication of the presence of mutagenic components in amylase, batch PPY32896, when tested under the conditions applied in this study, in the presence and absence of S-9.