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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 May 2009 - 04 June 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Active enzyme protein of Lipase, triacylglycerol (EC no. 232-619-9, CAS no. 9001-62-1, EC name: Lipase, triacylglycerol, Enzyme Class No.: 3.1.1.3)
Cas Number:
9001-62-1
Molecular formula:
Not applicable, see remarks
IUPAC Name:
Active enzyme protein of Lipase, triacylglycerol (EC no. 232-619-9, CAS no. 9001-62-1, EC name: Lipase, triacylglycerol, Enzyme Class No.: 3.1.1.3)
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:
- Substance type: UVCB
- Physical state: translucent tan liquid

Method

Target gene:
Histidine or tryptophan locus in the genome of five strains of bacteria
Species / strain
Species / strain / cell type:
bacteria, other: Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2uvrA
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver, S-9 mix
Test concentrations with justification for top dose:
Preliminary test: Concentrations tested were 3, 10, 33, 100, 333, 1000, 2500 and 5000 ug total protein/plate
Experiment 1: Eight concentrations of the test item (3, 10, 33, 100, 333, 1000, 2500 and 5000 ug total protein/plate)
Experiment 2: Eight concentrations of the test item (3, 10, 33, 100, 333, 1000, 2500 and 5000 ug total protein/plate)
Vehicle / solvent:
sterile deionised water
Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
sterile deionised water
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and experimental conditions:
The study describes experiments performed to assess the effect of lipase in amino acid dependent strains of Salmonella typhimurium and Escherichia coli capable of detecting both induced frame-shift (TA1537 and TA98) and base-pair substitution mutations (TA1535, TA100, and WP2uvrA). The test system is a reverse mutation of amino acid dependent bacterial strains. The potential of lipase to induce gene mutations was tested in the plate incorporation test (experiment I) and the pre-incubation test (experiment II).
DURATION
- Exposure duration, pre-incubation: 1 hour
- Incubation time (selective incubation): at least 48 hours
DETERMINATION OF CYTOTOXICITY
- Method: Reduction in number of colonies and clearance of bacterial background lawn
Evaluation criteria:
A test item is considered as a mutagen if all of the following criteria are met:
1) A biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and WP2uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed (Holstein et al. 1979)
2) A dose response dependant increase is considered biologically relevant if the threshold is exceeded at more than one concentration (De Serres and Shelby, 1979)
3) The increases are reproducible between replicate plates
4) An increase exceeding the threshold at only one concentration is judged as biologically relevant reproduced in an independent second study
Statistics:
Not performed.

Results and discussion

Test results
Key result
Species / strain:
bacteria, other: Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2uvrA
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:
The test item lipase was considered to be non-mutagenic under the conditions of this test.

Applicant's summary and conclusion

Conclusions:
Lipase is not mutagenic in the Ames assay in both the presence and absence of metabolic activation
Executive summary:

The objective of this assay was to assess the potential of lipase to induce gene mutations (frame-shift and base-pair) in four strains of Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvrA. Two experimental procedures were used – plate incorporation test (experiment I) and pre-incubation test (experiment II). The test material was tested both in the presence and absence of a metabolic activation system (Aroclor 1254-induced rat liver; S-9 mix). Experiment I was performed for dose selection with dose ranging from 3 to 5000 µg/plate. Since no toxicity was noted in Experiment I, eight dose concentrations ranging from 3 to 5000 µg/plate were selected for Experiment II. Triplicate plates were used at each test point. All dose levels were expressed in terms of total protein (TP). The highest dose level tested (5000 µg TP/plate) is the maximum required by the OECD guideline. Sterile deionized water was the vehicle used. Positive controls for assays without metabolic activation were sodium azide, 4-nitro-o-phenylene-diamine and methyl methane sulfonate. Positive control for assays with metabolic activation was 2-aminoanthracene. The study was conducted in accordance with OECD guideline No. 471 (1997) and complied with all standard GLP.

Lipase was not toxic to the test bacteria up to and including the highest dose level (5000 µg TP/plate) in both absence and presence of S-9 mix. No biologically significant increases in the number of revertant colonies were observed at any dose level tested in both presence and absence of S-9 mix. Distinct increases in revertant colonies were noted in all positive control assays.

 

Lipase is to be classified as “Non-Mutagenic” in this assay.