Nuclease, deoxyribo-

Genetic toxicity: in vitro

Administrative data

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

Justification for type of information:

Due to the similarity between the two enzymes and the fact that they belong to the same enzyme-sub class, similar results are expected for deoxyribonuclease.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

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

Test material

Test material information

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)

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:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Constituent 4

Reference substance name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available.

IUPAC Name:

Inorganic salts as a 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.

IUPAC Name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Test material form:

solid: particulate/powder

Details on test material:

- Lot/batch No.: PPW 2771
- Expiration date of the lot/batch: 1999-04-03

Method

Target gene:

HGPRT (6-thioguanine resistance)

Species / strain

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.

Controls

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

Results and discussion

Test results

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.

Applicant's summary and conclusion

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.