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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30-05-1990 to 16-11-1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
May 26th 1983
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent

Method

Target gene:
Histidine and tryptophan locus in the genome of four strains of bacteria
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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: 0.625, 1.25, 2.5, 5, 10 mg/mL.
The OECD 471 guideline from 1983 did not indicate a maximum dose to be used in the Ames test. 10 mg/mL was used as the maximum concentration in the laboratory.
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:
9-aminoacridine
2-nitrofluorene
other: 2-Aminoanthracene, N-Methyl-N'-Nitro-Nitrosoguanidine,
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°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.

Evaluation criteria:
For Salmonella typhimurium strain TA 1535, TA 1537 and TA 98 at least a doubling of the mean control value and a dose related response was looked for. At high dose levels this may be reverted because of toxicity to the bacteria. For TA 100 a 50% reproducible increase over control value is considered as indicative of a mutagenic effect .
Statistics:
N/A.

Results and discussion

Test resultsopen allclose all
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
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:
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

Applicant's summary and conclusion

Conclusions:
The results of the bacterial mutagenicity tests give no indication of mutagenic activity of beta-glucosidase batch DCN 0005 when tested in the presence or absence of the S-9 metabolic system. All results were confirmed by conducting two independent experiments.
Executive summary:

Beta-glucosidase batch DCN 0005 was examined for mutagenic activity using Salmonella typhimurium strains TA 1535, TA 100, TA 1537 and TA 98. A liquid culture assay was applied. Bacteria were exposed to 5 doses (0.625, 1.25, 2.5, 5, 10 mg/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 number of revertants to prototrophy and viable cells were estimated. The test was conducted in the presence and absence of metabolic activation - a liver preparation from male rats, pre-treated with Aroclor 1254, and the co-factors required for mixed function oxidase activity (S-9 mix). The sensitivity of the individual bacterial strains was confirmed by significant increases in number of revertant colonies induced in similar conditions by diagnostic mutagens. All results were confirmed by conducting two independent experiments. No dose-related and reproducible increases in revertants to prototrophy were obtained with any of the bacterial strains exposed to Novozym 188, either in the presence or absence of S-9 mix.

It was concluded that the results of the experiments give no indication of mutagenic activity of beta-glucosidase batch DCN 0005 in the presence or absence of metabolic activation.