Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Lipase has been investigated in several in vitro test systems, the Ames test, the in vitro chromosome aberration test, in a cultured human peripheral blood lymphocyte micronucleus assay and the in vitro mouse lymphoma test.

All tests have been performed according to current OECD guidelines, and in compliance with GLP.

No evidence for genetic toxicity was observed in any of the tests.

Link to relevant study records

Referenceopen allclose all

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
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
Target gene:
Histidine or tryptophan locus in the genome of five strains of bacteria
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.
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.
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.
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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 May 2009 - 20 July 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Test concentrations with justification for top dose:
Range finder: Concentrations tested were 32.5, 56.8, 99.5, 174.1, 304.6, 533.1. 932.9, 1632.7, 2857.1 and 5000 ug total protein/mL
Experiment 1: concentrations of the test item were 1632.7, 2857.1 and 5000 ug total protein/mL
Experiment 2: concentrations of the test item were 1632.7, 2857.1 and 5000 ug total protein/mL
Vehicle / solvent:
Sterile deionised water
Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
Negative solvent / vehicle controls:
yes
Remarks:
sterile deionised water
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Test item was accurately measured and dissolved in deionised water just before dosing the test system. The final conc of deionised water in the culture medium was 10% v/v. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 45 mOsm. Ten concentrations of lipase were used in the preliminary assay for the selection of dose levels in the main studies. Three doses were then selected for the two main assays ideally with the highest dose level inducing a toxic effect (50% reduction in mitotic index). In the absence of cytotoxicity, the highest dose selected would be 5000 µg total protein/mL (equivalent to 3834 µg TOS/mL) as recommended by the OECD guideline.

The used metabolic activation system was Aroclor 1254-induced rat liver homogenate, S-9 mix. The positive controls were: Ethylmethane sulfonate (EMS) for assays without metabolic activation (Acros, Batch No. A0259466) and Cyclophosphamide (CPA) for assays with metabolic activation (Aldrich Chemie, Batch No.097K1311).

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at 3 dose levels, together with negative control (solvent control: Deionised water 10%) and positive controls.  The treatment conditions used for the study were: In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I & II) after start of treatment with the test item. Mitosis was arrested by addition of Colcemid (0.2 µg/mL) 3 hours before the required harvest time.
Evaluation criteria:
Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the positive controls in Experiment I with metabolic activation, where only 50 metaphase plates were scored. Only metaphases with 46 ± 1 centromer regions were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Key result
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the preliminary assay, no significant cytotoxicity was noted up to the highest dose tested 5000 µg total protein/mL. Therefore, the following three doses were selected for both Experiment I and II: 1632.7, 2857.1 and 5000 µg total protein/mL.
In this study, in both experiment I and II, and in the absence and presence of S-9 mix, no cytotoxicity was observed and no biologically relevant increase in the number of cells with structural chromosomal aberrations was observed. No evidence of an increase in polyploidy metaphases was recorded with the test article. Significant increases in structural chromosomal aberrations were found with the positive controls, ethylmethane sulfonate for assays without S-9 mix and cyclophosphamide for assays with S-9 mix.

Conclusion: Non-clastogenic to human lymphocytes in vitro.
Conclusions:
Lipase is not clastogenic in the in vitro cytogenetic test using cultured human lymphocytes in both the presence and absence of metabolic activation.
Executive summary:

The objective of this assay was to investigate the potential of lipase to induce numerical and/or structural changes in the chromosome of mammalian systems (i.e., human peripheral lymphocytes) in both the presence and absence of metabolic activation (Aroclor 1254-induced rat liver; S-9 mix). This assay was conducted in accordance with OECD guideline No. 473 and complied with GLP. 

A preliminary cytotoxicity test was performed as a dose range finder using 10 concentrations of lipase and a solvent and a positive control. Doses selected for the main tests (Experiment I and II) were based on cytotoxicity, which was characterized by the percentage of mitotic suppression in comparison to controls. In the preliminary assay, no significant cytotoxicity was noted up to the highest dose tested 5000 µg total protein/mL. Therefore, the following three doses were selected for both Experiment I and II: 1632.7, 2857.1 and 5000 µg total protein/mL. The 5000 µg/mL is the highest dose recommended by the OECD guideline. 

In Experiment I, the exposure period was 4 hours for assays with and without S-9 mix. In Experiment II, the exposure period was 4 hours for assays with S-9 mix and 22 hours for assays without S-9 mix. In both experiments, colcemid was added to the cultures three hours prior to harvesting. Cells were then harvested, collected, suspended, fixed, and evaluated.

In this study, in both experiment I and II, and in the absence and presence of S-9 mix, no significant cytotoxicity was observed and no biologically relevant increase in the number of cells with structural chromosomal aberrations was observed. No evidence of an increase in polyploidy metaphases was recorded with the test article. Significant increases in structural chromosomal aberrations were found with the positive controls, ethylmethane sulfonate for assays without S-9 mix and cyclophosphamide for assays with S-9 mix.

 

Lipase is to be classified as “Non-Clastogenic” in this in vitro cytogenetic test using cultured human lymphocytes cells both in the presence and absence of metabolic activation.

 

GHS Classification: Not classified

 

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 June 1989 - 11 October 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT (6-thioguanine resistance)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fishers medium (10% and 20% horse serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
The highest concentration tested was 5000 µg/mL, tested as supplied (equivalent to 4585 ug enzyme concentrated dry matter/mL).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure is in aqueous solutions.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline-1-oxide (without S-9) and benzo(a)pyrene (with S-9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium, growth suspension. Selection phase was performed in microtitre plates.

DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium): With exception of experiment 1 treatments in the absence of S-9 (where cultures were maintained for eight days) cultures were maintained for 7 days.
- Fixation time (start of exposure up to fixation or harvest of cells): At least 7 days after treatment.

SELECTION AGENT (mutation assays): 6-TG

NUMBER OF REPLICATIONS: duplicate

DETERMINATION OF CYTOTOXICITY
- Method: Cell density by counting viable cells, expressed as percentage relative survival (RS%)
Evaluation criteria:
A test article was considered positive if:
- The assay was valid, and
- Significant induced mutation (i.e. the lower 95 percentile of a treated culture exceeded the upper 95 percentile of a control culture) occurred at consecutive doses in at least one experiment, and
- Dose-related increases in mutation could be confirmed by regression analysis in both experiments.
Statistics:
The mutation frequency was evaluated statistically by using logarithmic transformation of the variances of the number of clones observed on viability and mutation plates as described by E.E. Furth et al., Anal Biochem 110: 1-8, 1981
Key result
Species / strain:
mouse lymphoma L5178Y cells
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:
No Lipase treatment in the presence of S-9 in either experiment, or in the absence of S-9 in experiment 1, resulted in a statistically significant increase in mutation frequency. Experiment 2 treatments in the absence of S-9, did result in a small but statistically significant increase in mutation frequency. However, this significant increase was observed only at the lower dose plated for determination of 6-thioguanine resistance, and furthermore, showed no dose-correlation by linear-regression analysis. The data therefore cannot be considered evidence of mutation induction at the HGPRT locus of LS178Y mouse cells.
Conclusions:
It was concluded that Lipase had no mutagenic activity in the present test system.
Executive summary:

Lipase was assayed for its ability to induce mutation at the HGPRT locus (6-thioguanine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of two independent experiments, each conducted in the absence and presence of metabolic activation (S-9 mix).

Following a wide range of treatments, separated by half-log intervals and reaching 5000 µg/mL (equivalent to 4585 ug enzyme concentrated dry matter/mL), cells survived this dose of 5000 µg/mL (93% survival) in the absence and 500 µg/mL (11% survival) in the presence of S-9. This, together with the next three lower doses without S-9 and the next five lower doses with S-9, were plated for viability and 6-thioguanine resistance seven days after treatment (with the exception of experiment 1 treatments in the absence of S-9, plated after eight days). In the second experiment a narrower dose range was used to maximise the chance of detecting any lose related effects. The top doses plated in this experiment were 5000 µg/mL in the absence and 500 µg/mL in the presence of S-9, which yielded 104% and 5% survival, respectively.

Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S-9. Mutation frequencies in negative control cultures fell within normal ranges, and statistically significant increases in mutation were induced by the positive control chemicals 4-nitroquinoline-1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore, the study was accepted as valid.

No lipase treatment in the presence of S-9 in either experiment, or in the absence of S-9 in experiment 1, resulted in a statistically significant increase in mutation frequency. Experiment 2 treatments in the absence of S-9, did result in a small but statistically significant increase in mutation frequency. However, this significant increase was observed only at the lower dose plated for determination of 6-thioguanine resistance, and furthermore, showed no dose-correlation by linear-regression analysis. The data therefore cannot be considered evidence of mutation induction at the HGPRT locus of LS178Y mouse cells.

It was concluded that lipase had no mutagenic activity in this test system.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov. 2, 2006 - Jan. 3, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: Draft OECD guideline 487, adopted 22 July 2010
Deviations:
no
Principles of method if other than guideline:
Lipase was tested for induction of micronuclei using human lymphocyte cultures prepared from heparinised whole blood cultures pooled from two male donors. Mitotic division of the lymphocytes was stimulated, by adding phytohaemagglutinin (PHA) to the cultures for 24 hours in first experiment and 48 hours in second experiment before treatment.
Sets of duplicate cultures were treated with the solvent (sterile purified water), test chemical or positive controls in the presence and absence of a metabolic activation system (S-9). In both experiments treatment in the absence of S-9 was for 20 hours followed by a 28-hour recovery period prior to harvest. Treatment in the presence of S-9 was for 3 hours (pulse treatment) followed by a 45-hour recovery period prior to harvest.
By the addition of the actin inhibitor cytochalasin-B about 28 hours prior to harvest cytokinesis (cell division) was blocked resulting in the formation of binucleate cells (the cytokinesis-block methodology).
4-Nitroquinoline 1-oxide and Vinblastine were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of liver S-9. Cyclophosphamide was employed as a clastogenic positive control chemical in the presence of liver S-9. Treatments with lipase covered a broad range of doses, separated by narrow intervals, where the highest dose level used was 5000 ug/mL. Cells were harvested by repeated centrifugation, hypotonic treatment and fixation. Slides were prepared and cells were stained with Giemsa.
Micronuclei were counted in binucleate cells at three consecutive dose levels, selected by evaluating the effect of lipase on the replication index (RI) as a measure for cytotoxicity.
A total of 1000 cells per dose level (500 from each replicate culture) from the selected lipase treatments and solvent controls were scored blind for micronuclei in binucleated cells. The proportion of cells with micronuclei for each treatment condition were compared with the proportion in solvent controls by using Fisher's exact test.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: Primary cells from human blood
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver homogenate, S-9 mix
Test concentrations with justification for top dose:
Highest concentration tested was 5000 µg/mL (weighed out as received) (equivalent to 595 µg enzyme concentrate dry matter/mL) and dilutions hereof.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Purified water
- Justification for choice of solvent/vehicle: Substance is water-soluble and any human exposure will be in aqueous solutions.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-Nitroquinoline 1-oxide (NQO) and Vinblastine (VIN) in the absence of rat liver S-9, Cyclophosphamide (CPA) in the presence of S-9.
Details on test system and experimental conditions:
METHOD OF APPLICATION: Whole blood cultures were established in HEPES-buffered RPMI medium containing 20% (v/v) heat inactivated foetal calf serum, 50 μg/mL gentamycin and 2% of the mitogen Phytohaemagglutinin (PHA). These cultures were incubated at approx. 37°C for 24 hrs (Exp. 1) or 48 hrs (Exp. 2) before treatment with test article. Sets of duplicate cultures were treated with the solvent (sterile purified water), test chemical or positive controls in the presence and absence of a metabolic activation system prepared from a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals.
In both experiments treatment in the absence of S-9 was for 20 hours, followed by a 28-hour recovery period prior to harvest. Treatment in the presence of S-9 was for 3 hours (pulse treatment) followed by a 45-hour recovery period prior to harvest. By the addition of the actin inhibitor cytochalasin-B about 28 hours prior to harvest cytokinesis (cell division) was blocked resulting in the formation of binucleate cells (the cytokinesis-block methodology). 4-Nitroquinoline 1-oxide and Vinblastine were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of liver S-9. Cyclophosphamide was employed as a clastogenic positive control chemical in the presence of liver S-9.
Treatments with the test substance covered a broad range of doses, separated by narrow intervals, where the highest dose level used was 5000 ug/mL. Cells were harvested by repeated centrifugation, hypotonic treatment and fixation. Slides were prepared and cells were stained with Giemsa.
Micronuclei were counted in binucleate cells at three consecutive dose levels, selected by evaluating the effect of the test substance on the replication index (RI) as a measure for cytotoxicity.
A total of 1000 cells per dose level (500 from each replicate culture) from the selected treatments and solvent controls were scored blind for micronuclei in binucleated cells.

NUMBER OF REPLICATIONS: Sets of duplicate cultures were exposed to the test substance.
Evaluation criteria:
A test article was considered positive if:
- the assay was valid, and
- significant increase in the frequency of micronucleated binucleate cells (MNBN) at one or more concentrations , and
- the incidence of MNBN cells exceeded the normal range in both replicates, and
- a concentration-related increase in the proportion of MNBN cells was observed.
Statistics:
The proportion of MNBN cells for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test. Probability values of p equal or less than 0.05 were accepted as significant.
Key result
Species / strain:
lymphocytes: from human blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight cytotoxicity (RI reduction 15 resp. 21%) was seen in both experiments in the presence of S-9 mix at the highest concentration 5000 µg/mL. No significant cytotoxicity was seen without S-9.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: no
- Water solubility: yes
- Precipitation: no
- Other confounding effects: none

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative controls were within the historical negative control ranges.
Conclusions:
Lipase did not show any clastogenic activity, neither in the presence or absence of S-9 mix, when tested in the present in vitro micronucleus assay.
Executive summary:

The clastogenic and aneugenic activity of lipase was investigated in cultured human peripheral blood lymphocytes by effects on the frequency of micronuclei. Division of the lymphocytes was stimulated by adding phytohaemagglutinin to the cultures.

In both experiments treatment in the absence of S-9 was for 20 hours followed by a 28-hour recovery period prior to harvest. Treatment in the presence of S-9 was for 3 hours (pulse treatment) followed by a 45-hour recovery period prior to harvest. By the addition of the actin inhibitor cytochalasin-B about 28 hours prior to harvest, cell division was blocked resulting in the formation of binucleate cells. 4-Nitroquinoline 1-oxide and Vinblastine were clastogenic and aneugenic positive controls in the absence of liver S-9, while Cyclophosphamide was positive control in the presence of liver S-9.

 

Treatments with the test substance covered a broad range of doses; the highest dose level was 5000 ug test item/mL (equivalent to 595 ug enzyme concentrate dry matter/mL).

 

The proportion of binucleate cells with micronuclei in all cultures of the vehicle controls was within the limits of the historical ranges. The positive controls induced statistically significant increases in the proportion of cells with micronuclei, demonstrating the sensitivity of the test procedure and the metabolic activity of the S-9 mix employed.

 

In the first experiment the highest concentration of lipase chosen for analysis, 5000 ug/mL in the absence of S-9 and 5000 ug/mL in the presence of S-9, induced approximately 0% and 21% reduction in replication index (RI) as a measure for cytotoxicity.

 

In the second experiment the highest concentration chosen for analysis, 5000 ug/mL in the absence and the presence of S-9, induced approximately 0% and 15% reduction in RI.

 

In both experiments treatment of cells with lipase in the absence and presence of metabolic activation (S-9) resulted in frequencies of micronucleated binucleate cells (MNBN), which were similar to those observed in concurrent vehicle control cultures for all concentrations analysed.

 

Lipase (batch PPW 26090) did not induce micronuclei in cultured human peripheral blood lymphocytes following either 20 hour or 3 hour treatment with concentrations up to 5000 ug/mL (equivalent to 595 ug enzyme concentrate dry matter/mL) in the absence and presence of a rat liver metabolic activation system (S-9) [20+28 hour -S-9, or 3+45 hour +S-9] where treatments commenced 24 or 48 hours following PHA (mitogen) stimulation.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The genetic toxicity of lipase has been investigated in the Ames assay, in the in vitro chromosome aberration assay, in the in vitro mammalian cell test looking at the induction of micronuclei and in the mouse lymphoma assay. All tests have been performed according to OECD and EC guidelines, and in compliance with GLP. No evidence for genetic toxicity was observed in any of the tests and thus it can be concluded that lipase is not mutagenic and does not induce chromosome aberration in the present test systems.

Justification for classification or non-classification

Due to the lack of genetic toxicity, lipase should not be classified.