Nuclease, deoxyribo-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: It was concluded that Deoxyribonuclease, batch PPW38822 is not mutagenic in histidine-requiring Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537), and tryptophan-requiring Escherichia coli strain (WP2uvrApKM101), when tested under the conditions of this study. These conditions included treatments at concentrations equivalent to 5885 μg enzymes concentrate dry matter/mL with and without the rat liver metabolic activation system (S9), and using a treat and plate assay.

Ames: It was concluded that deoxyribonuclease, batch PPW42035 did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2 uvrA pKM101) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 16667 μg enzymes concentrate dry matter/mL), in the absence and in the presence of a rat liver metabolic activation system (S-9) using a modified Treat and Plate methodology.

In-vitro micronucleus: It was concluded that Deoxyribonuclease, PPW38822 did not induce biologically relevant increases micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9). Concentrations were analysed up to 5885 μg enzyme concentrate dry matter/mL (3+21 hour –S-9treatment), or, to a suitable limit of cytotoxicity (3+21 hour +S-9 and 24+24 hour -S-9treatments).

In-vitro micronucleus: During the testing of deoxyribonuclease batch PPW42035 in micronuclei in cultured human peripheral blood lymphocytes, some osmolality shifts were noted. As it is known that marked shifts in osmolality can give rise to chromosome aberrations (and thereby micronuclei), these observations cast some doubt over the biological significance of the observed increases in MNBN cells observed. However, osmolality was not found to be a confounding factor as increased frequencies of MNBN cells were observed at concentrations below where any marked osmolality shifts were apparent. It was concluded that deoxyribonuclease batch PPW42035 induced micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of an induced rat liver metabolic activation system (S-9) in two independently conducted experiments. Concentrations were tested up to 8333 μg enzyme concentrate dry matter/mL), a recommended regulatory maximum concentration for the in vitro micronucleus assay.

In-vivo micronucleus: It was concluded that deoxyribonuclease, batch U8AGS, formulated in phosphate buffered saline did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male mice when dosed intravenously with up to 400 mg/kg/day (an estimate of the maximum tolerated dose for this study), under the experimental conditions employed.

Link to relevant study records

Reference

Reference 1

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04-01-2016 to 25-04-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan locus in the genome of five strains of bacteria

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

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 were used (184, 368, 736, 1471, 2943, 5885 μg enzyme concentrate dry matter/mL).

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: Acridine mutagen (ICR-191), 2-Aminoanthracene (2-AA)

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 ± 2°C for 3 hours (treat and plate).
- Incubation time (selective incubation): about 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count.

Evaluation criteria:

The test substance is considered mutagenic when it induces at least a doubling in the mean number of revertant colonies per plate, compared to the appertaining solvent control. The response must be dose related, reproducible between replicates and experiments, and independent of growth stimulation of non-revertant bacteria. The response can be observed in one or more of the bacterial strains and in the absence or presence of S9 mix. 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:

Not performed.

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

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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, solvent controls.

Conclusions:

It was concluded that Deoxyribonuclease, batch PPW38822 is not mutagenic in histidine-requiring Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537), and tryptophan-requiring Escherichia coli strain (WP2uvrApKM101), when tested under the conditions of this study. These conditions included treatments at concentrations up to 5885 μg enzymes concentrate dry matter/mL with and without the rat liver metabolic activation system (S9), and using a treat and plate assay.

Executive summary:

Deoxyribonuclease, batch PPW38822 was examined for mutagenic activity in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium strain TA1535, TA100, TA1537, TA98 and Escherichia coli WP2uvrApKM101. Crude enzyme preparations, like the present test substance contain free amino acids histidine and tryptophan, most often in an amount, which exceed the critical concentration for incorporation in the direct standard assay (plate incorporation method). To overcome this problem, all strains were exposed to the test substance in liquid culture in a treat and plate method. Bacteria were exposed to six doses of the test substance in a phosphate buffered nutrient broth for 3 hours with 5885 μg enzymes concentrate dry matter/mL as the highest concentration. After incubation, the test substance was removed by centrifugation prior to plating. The study was conducted without and with the metabolic activation system (S9 mix). The metabolic activation system was a liver preparation from male rats pre-treated with Aroclor 1254 (S9), added with co-factors required for mixed function oxidase activity. Two complete and independent experiments were conducted before any conclusions were made.

Deoxyribonuclease, batch PPW38822 exposure did not increase the number of revertant colonies of the Salmonella and E. coli strains, that meet the criteria for a positive or equivocal response.

Based on the results obtained in this study it was concluded that Deoxyribonuclease, batch PPW38822, is not mutagenic when tested under the conditions applied in this bacterial reverse mutation test.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-03-2017 to 03-08-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Principles of method if other than guideline:

The top dose used was 16667 μg enzymes concentrate dry matter/mL to ensure the highest possible exposure.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan locus in the genome of five strains of bacteria.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

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.
Justification for top dose: The top dose used was 10000 TOS μg/mL (equivalent to 16667 μg enzymes concentrate dry matter/mL), which is twice the dose recommended by the guideline. The reason was to increase the amount of active enzyme protein and see if any effects are observed.
Experiment 1: 53, 167, 533, 1667, 5333, 16667 μg enzymes concentrate dry matter/mL
Experiment 2: 171, 427, 1067, 2667, 6667, 16667 μg enzymes concentrate dry matter/mL

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

other: N-methyl-N'-nitro- N-nitrosoguanidine, ICR-191 mutagen (ICR-191), 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 10^8 to 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: Following incubation, the mutation plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted electronically using a Sorcerer Colony Counter.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥2-fold (in strains TA98, TA100 and WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control values.
2. The positive trend/effects described above were reproducible.
The test article was considered as positive in this assay if the above criteria were met.
The test article was considered as negative in this assay if the above criteria were not met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and between experiments.

Statistics:

Not performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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:

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes, vehicle controls.

Conclusions:

It was concluded that deoxyribonuclease, batch PPW42035 did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2 uvrA pKM101) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 16667 μg enzymes concentrate dry matter/mL), in the absence and in the presence of a rat liver metabolic activation system (S-9) using a modified Treat and Plate methodology.

Executive summary:

Deoxyribonuclease, batch PPW42035 was assayed for mutation in four histidine requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2 uvrA pKM101) of Escherichia coli, both in the absence and presence of metabolic activation (S-9) in two separate experiments. A 'treat and plate' procedure was used for all treatments in this study as deoxyribonuclease, batch PPW42035 may contain free amino acids i.a. histidine and tryptophan (which may cause artefacts through growth stimulation in a standard plate-incorporation test). All Deoxyribonuclease, batch PPW42035 treatments in this study were performed using formulations prepared in water for irrigation (purified water).

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of deoxyribonuclease, batch PPW42035 at 53, 167, 533, 1667, 5333, 16667 μg enzymes concentrate dry matter/mL, the maximum concentration being in excess of the required regulatory limit level of 5000 μg/mL. Following these treatments, evidence of toxicity was observed at 5333 μg enzyme concentrate dry matter/mL and above in strains TA100, TA1535 and TA1537 in the absence of S-9 and in strain TA100 in the presence of S-9, and at 16667 μg enzymes concentrate dry matter/mL in strain TA1537 in the presence of S-9.

Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 16667 μg enzymes concentrate dry matter/mL was retained for all strains. Narrowed concentration intervals were employed covering the range of 171-16667 μg enzymes concentrate dry matter/mL, in order to examine more closely those concentrations of Deoxyribonuclease, batch PPW42035 approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. Following these treatments, evidence of toxicity was observed at 6667 μg enzymes concentrate dry matter/mL and above in strains TA100 and TA1535 in the absence and presence of S-9 and in strain TA1537 at 16667 μg enzymes concentrate dry matter/mL in the absence and presence of S-9. The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

Following Deoxyribonuclease, batch PPW42035 treatments of all the test strains in the absence and presence of S-9, no notable, concentration-related and reproducible increases in revertant numbers were observed that were ≥2-fold (in strains TA98, TA100 and WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any mutagenic activity in this assay system.

It was concluded that Deoxyribonuclease, batch PPW42035 did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2 uvrA pKM101) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 16667 μg enzymes concentrate dry matter/mL (a maximum concentration in excess of that required by current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9) using a modified Treat and Plate methodology.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18-12-2015 to 16-05-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2014

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: cultured human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

Blood of two male donors.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

N/A

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The highest concentration selected for micronucleus analysis following all treatment conditions was the highest concentration tested (5000 μg TOS/mL, equivalent to 5885 μg enzyme concentrate dry matter/mL), a recommended maximum concentration for in vitro micronucleus studies (OECD, 2010). Other concentrations include 10, 100, and 1000 μg TOS/mL (equivalent to 12, 118, 1177 μg enzyme concentrate dry matter/mL) for 3+21 hour -S-9 treatment; 100, 250, 1000, 3500 μg TOS/mL (equivalent to 118, 294, 1177, 4120 μg enzyme concentrate dry matter/mL) for 3+21 hour +S-9 treatment; 1, 5, 20, 30, 40, 50 μg TOS/mL (equivalent to 1.2, 5.9, 24, 35, 47, 59 μg enzyme concentrate dry matter/mL) for 24+24 hour -S-9 treatment.

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Details on test system and experimental conditions:

METHOD OF APPLICATION: Cells were placed in HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin. The mitogen Phytohaemagglutinin was included in theculture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37±1°C for approximately 48 hours and rocked continuously.

DURATION
- Preincubation period: Blood cultures were incubated at 37±1°C for approximately 48 hours and rocked continuously.
- Exposure duration:
Experiment 1: 3+21h without S-9; 3+21h with S-9; 24+24h without S-9.
Experiment 2:3+21h with S-9.
- Expression time (cells in growth medium): 1.5-2.0 cell cycles (approximately 24 hours)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 or 48 hours

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): Acridine Orange

NUMBER OF REPLICATIONS: A, B. Sets of duplicate cultures were exposed to the test substance, in at least two independent experiments.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Lymphocytes were kept in fixative at 2-8°C prior to slide preparation for a minimum of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged and resuspended in a minimal amount of fresh fixative. Cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were stained by immersion in 125 μg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 seconds, washed with PBS (with agitation) for a few seconds before transfer and immersion in a second container of PBS for approximately 10 minutes. Slides were air-dried and stored protected from light at room temperature prior to analysis.

NUMBER OF CELLS EVALUATED: 2000 per concetration

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Binucleate cells were only included in the analysis if all of the following criteria were
met:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.

A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index. S-9 mix or KCl (0.5 mL per culture) was added appropriately. Cultures were treated with the test article, vehicle (1 mL per culture). Positive control treatments were not included. The final culture volume was 10 mL. Cultures were incubated at 37±1°C for the designated exposure time. The highest concentration for micronucleus analysis should typically be one at which approximately 55±5% reduction in RI has occurred or should be the highest concentration tested.
- Any supplementary information relevant to cytotoxicity: Cytotoxicity (%) is expressed as (100 – Relative Replication Index).

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:

1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed.

The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.

Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result

Statistics:

The proportion of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test. A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p≤0.05 were accepted as significant.

Key result

Species / strain:

lymphocytes: cultured human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No marked changes in osmolality (shifts of greater than 50 mOsm/kg) or pH (shifts of greater than 1 pH unit) were observed at the highest concentration tested (5000 μg/mL) as compared to the concurrent vehicle controls.
- Evaporation from medium: N/A
- Water solubility: Enzymes are water soluble.
- Precipitation: Enzymes are water soluble.
- Definition of acceptable cells for analysis: The cytoplasm remained essentially intact, and the daughter nuclei were of approximately equal size.

RANGE-FINDING/SCREENING STUDIES: Cytotoxicity range finding was conducted.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: Yes

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: Not statistically significant
- Indication whether binucleate or mononucleate where appropriate: Yes.

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: The proportion of micronucleated binucleate cells in the vehicle cultures fell within current 95th percentile of the observed historical vehicle control (normal) ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Slides from the cytotoxicity Range-Finder Experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) was determined. Cytotoxicity (%) is expressed as (100 – Relative RI). A selection of random fields was observed from enough treatments to determine whether chemically induced cell cycle delay or cytotoxicity had occurred.

Conclusions:

It was concluded that Deoxyribonuclease, PPW38822 did not induce biologically relevant increases micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9). Concentrations were analysed up to 5885 μg enzyme concentrate dry matter/mL (3+21 hour –S-9treatment), or, to a suitable limit of cytotoxicity (3+21 hour +S-9 and 24+24 hour -S-9treatments).

Executive summary:

Deoxyribonuclease, PPW38822 was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two male donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats.

The test article was formulated in water for irrigation (purified water). The highest concentrations tested in the Micronucleus Experiment were determined following a preliminary cytotoxicity Range-Finder Experiment. Treatments were conducted (as detailed in the following summary table) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of Deoxyribonuclease, PPW38822 on the replication index (RI). Micronuclei were analysed at four or six concentrations: 12, 118, 1177, 5885 μg enzyme concentrate dry matter/mL for 3+21 hour -S-9 treatment; 118, 294, 1177, 4120 μg enzyme concentrate dry matter/mL for 3+21 hour +S-9 treatment; 1.2, 5.9, 24, 35, 47, 59 μg enzyme concentrate dry matter/mL for 24+24 hour -S-9 treatment.

Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in the vehicle cultures fell within the current 95th percentile of the observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment.

All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei. All acceptance criteria were considered met and the study was therefore accepted as valid.

Pulse (3+21 hour) treatment of cells with Deoxyribonuclease, PPW38822 in the absence and presence of S-9 resulted in frequencies MNBN cells which were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for all concentrations analysed. The MNBN cell frequency of all bar a single treated culture fell within the 95th percentile of the normal ranges. These data indicated no adverse effect of Deoxyribonuclease, PPW38822 treatment. Following extended 24+24 hour treatment in the absence of S-9, a small but statistically significant (p≤0.01) increase in MNBN cells was observed at the highest concentration analysed (59 μg enzyme concentrate dry matter/mL, inducing 50% cytotoxicity). However, only one of the two replicate cultures exhibited a MNBN cell value that exceeded the normal range, and the increase was small (mean MNBN cell frequency was 1.05% compared to 0.4% for the concurrent vehicle control and normal range of 0.1 to 0.8% MNBN cells). The MNBN cell values for all other Deoxyribonuclease, PPW38822 treated cultures (all lower concentrations analysed including concentrations of 47 and 35 μg enzyme concentrate dry matter/mL, inducing 49% and 51% cytotoxicity respectively) fell within normal values. As such, this isolated statistical increase was not considered of biological importance

It was concluded that Deoxyribonuclease, PPW38822 did not induce biologically relevant increases micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9). Concentrations were analysed up to 5885 μg enzyme concentrate dry matter/mL (3+21 hour –S-9 treatment), or, to a suitable limit of cytotoxicity (3+21 hour +S-9 and 24+24 hour -S-9 treatments).

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

14-11-2017 to 03-07-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

no

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: cultured human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

Blood from two healthy male volunteers.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

N/A

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The highest concentration selected for micronucleus analysis following all treatment conditions was the highest concentration tested (5000 μg TOS/mL, equivalent to 8333 μg enzyme concentrate dry matter/mL), a recommended maximum concentration for in vitro micronucleus studies (OECD, 2010). Other concentrations for Experiment 1: 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 μg TOS/mL (equivalent to 833, 1667, 2500, 4167, 5000, 5833, 6667, 7500 μg enzyme concentrate dry matter/mL); and Experiment 2: 100, 250, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 μg TOS/mL (equivalent to 167, 417, 833, 1667, 2500, 4167, 5000, 5833, 6667, 7500 μg enzyme concentrate dry matter/mL).

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Details on test system and experimental conditions:

METHOD OF APPLICATION: Cells were placed in HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin. The mitogen Phytohaemagglutinin was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37±1°C for approximately 48 hours and rocked continuously.

DURATION
- Preincubation period: Blood cultures were incubated at 37±1°C for approximately 48 hours and rocked continuously.
- Exposure and revocery duration:
Experiment 1: 3+21h without S-9; 3+21h with S-9; 24+24h without S-9.
Experiment 2: 3+21h without S-9; 3+21h with S-9; 24+24h without S-9.
- Fixation time (start of exposure up to fixation or harvest of cells): Cultures were harvested 24 or 48 hours after the initiation of treatment.

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): Acridine Orange in phosphate buffered saline (PBS)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Slides from the highest selected concentration and three (Experiment 1) or five (Experiment 2) lower concentrations were taken for microscopic analysis, such that a range of cytotoxicity from maximum to little or none was covered, where appropriate.

NUMBER OF CELLS EVALUATED: 1000 per concetration, 2000 for the vehicle

DETERMINATION OF CYTOTOXICITY: Method: Cytotoxicity in % was expressed as 100-Replication Index (RI)..

Evaluation criteria:

Not specified

Statistics:

The proportion of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test. Probability values of p≤0.05 were accepted as significant.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: For Experiment 1: Assessment of post treatment medium for Osmolality and pH from the highest three concentrations tested (6667, 7500 and 8333 μg enzyme concentrate dry matter/mL) under each treatment condition indicated marked positive shifts in Osmolality (shifts of greater than 50 mOsm/kg) as compared to concurrent vehicle control values. For experiment 2: Some osmolality shifts were noted. However, osmolality was not found be a confounding factor as increased frequencies of MNBN cells were observed at concentrations below where any marked osmolality shifts were apparent.
- Evaporation from medium: N/A
- Water solubility: Enzymes are water soluble.

RANGE-FINDING/SCREENING STUDIES: Yes

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: Yes

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: Statistically significant
- Indication whether binucleate or mononucleate where appropriate: Yes.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity was assessed by calculating the replication index (RI) in test article-treated cultures, relative to the concurrent vehicle control values.

Conclusions:

During the testing of deoxyribonuclease batch PPW42035 in micronuclei in cultured human peripheral blood lymphocytes, some osmolality shifts were noted. As it is known that marked shifts in osmolality can give rise to chromosome aberrations (and thereby micronuclei), these observations cast some doubt over the biological significance of the observed increases in MNBN cells observed. However, osmolality was not found to be a confounding factor as increased frequencies of MNBN cells were observed at concentrations below where any marked osmolality shifts were apparent.
It was concluded that deoxyribonuclease batch PPW42035 induced micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of an induced rat liver metabolic activation system (S-9) in two independently conducted experiments. Concentrations were tested up to 8333 μg enzyme concentrate dry matter/mL), a recommended regulatory maximum concentration for the in vitro micronucleus assay.

Executive summary:

The objective of this study was to evaluate the clastogenic and aneugenic potential of deoxyribonuclease, batch PPW42035 by examining its effects on the frequency of micronuclei in cultured human peripheral blood lymphocytes treated in the absence and presence of a rat liver metabolising system (S-9).

Micronucleus Experiment 1:

Binucleate cells were analysed in cultures treated at 3333, 5000, 6667 and 8333 μg enzyme concentrate dry matter/mL (3+21 hours in the absence and presence of S-9) and at 833, 3333, 5833 and 8333 μg enzyme concentrate dry matter/mL (24+24 hours in the absence of S-9) for micronuclei. The highest concentration analysed for micronuclei, 8333 μg enzyme concentrate dry matter/mL, induced 16% (3+21 hours in the absence of S-9), 19% (3+21 hours in the presence of S-9) and 55% (24+24 hours in the absence of S-9) cytotoxicity. Treatment of cells with deoxyribonuclease, batch PPW42035 in the absence and presence of a rat liver metabolic activation system (S-9) resulted in frequencies of micronucleated binucleate (MNBN) cells which were significantly (p≤0.05) higher than those observed in concurrent vehicle controls for the majority of all concentrations analysed (all treatments). The MNBN cell frequency of the majority of all treated cultures exceeded the 95th percentile of the current observed historical vehicle control (normal) ranges. Although the magnitude of these increases was not large, they were consistent across treatment concentrations and treatment regimens. Assessment of post treatment medium for Osmolality and pH from the highest three concentrations tested (6667, 7500 and 8333 μg enzyme concentrate dry matter/mL) under each treatment condition indicated marked positive shifts in Osmolality (shifts of greater than 50 mOsm/kg) as compared to concurrent vehicle control values). As such, a second experimental trial was conducted with osmolality measurements taken at all concentrations tested and with a wide range of concentrations analysed where osmolality shifts ranged from little to marked.

Micronucleus Experiment 2:

Binucleate cells were analysed in cultures treated at 1667, 2500, 3333, 5000, 6667 and 8333 μg enzyme concentrate dry matter/mL (3+21 hours in the absence of S-9), at 833, 2500, 3333, 5000, 6667 and 8333 μg enzyme concentrate dry matter/mL (3+21 hours in the presence of S-9) or 417, 833, 1667, 3333, 5833 and 8333 μg enzyme concentrate dry matter/mL (24+24 hours in the absence of S-9) for micronuclei. The highest concentration analysed for micronuclei, 8333 μg enzyme concentrate dry matter/mL, induced 12% (3+21 hours in the absence of S-9), 15% (3+21 hours in the presence of S-9) and 48% (24+24 hours in the absence of S-9) cytotoxicity. Treatment of cells with deoxyribonuclease, batch PPW42035 in the absence and presence of S-9 resulted in frequencies of MNBN cells that were significantly (p≤0.05) higher than those observed in concurrent vehicle controls for all concentrations analysed (all treatments). The MNBN cell frequency of all deoxyribonuclease, batch PPW42035 treated cultures exhibited MNBN cell values that exceeded normal ranges. The most potent responses were observed following 24+24 hour -S-9 treatment. Concentrations analysed encompassed osmolality shifts ranging from either +101 mOsm/kg to +19 mOsm/kg (3+21 hour -S-9), +99 mOsm/kg to +15 mOsm/kg (3+21 hour +S-9) or +96 mOsm/kg to +5 mOsm/kg (3+21 hour -S-9). These data indicated a clear test article related effect on induction of MNBN cells.

As it is known that marked shifts in osmolality can give rise to chromosome aberrations (and thereby micronuclei), these observations cast some doubt over the biological significance of the observed increases in MNBN cells observed. However, osmolality was not found to be a confounding factor as increased frequencies of MNBN cells were observed at concentrations below where any marked osmolality shifts were apparent.

It was concluded that deoxyribonuclease batch PPW42035 induced micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of an Aroclor induced rat liver metabolic activation system (S-9) in two independently conducted experiments. Concentrations were tested up to 8333 μg enzyme concentrate dry matter/mL), a recommended regulatory maximum concentration for the in vitro micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Deoxyribonuclease, batch U8AGS was tested in the in vivo micronucleus test. Male mice were dosed intravenously with 400, 200 and 100 mg enzyme protein/kg/day. Based on the results obtained from this study, it was concluded that deoxyribonuclease, batch U8AGS, formulated in phosphate buffered saline did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male mice when dosed intravenously with up to 400 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Mouse Bone Marrow Micronucleus Assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11-04-2017 to 16-08-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

We understand that REACH requires that new testing of a substance involving vertebrate animals is only carried out as a last resort. A registrant shall submit a testing proposal and ECHA publishes all test proposals involving vertebrate animals, for endpoints specified in Annexes IX and X under REACH before the testing is carried out (REACH Art 40(2)). The test according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) is listed in Annex IX. Registrant made the study without a test proposal to ECHA because of the reasons stated below.

1. Uses in Section 3 and needs for global market
The substance in this dossier is an enzyme which should be used for uses stated in Section 3. The enzyme has been produced in Registrant’s site in EU, however the business decided to market the product first in Philippines. It is a new substance in accordance with the Philippine Inventory of Chemicals and Chemical Substances (PICCS). The enzyme was first notified for the low volume (less than 1 ton/year) with Small Quantity Importation (SQI) by Registrant’s business partner in Philippines. Along development of the market, there were needs for a Pre-Manufacture Pre-Importation Notification (PMPIN) for permit of higher tonnage import. Registrant conducted the in-vivo study for this purpose. Registrant’s business partner finally submitted PMPIN on January 24, 2018.

2. Other uses which are not in the scope of REACH
In addition, Registrant consider further development of uses of the enzyme within the food industry in EU and other countries. In EU, such an enzyme used as processing aid for food manufacture is in the scope of the EU regulatory framework, the Food Improvement Agents Package (FIAP) and a company must obtain approval from EFSA by submitting a dossier. According to the EFSA (CEF panel) guidance for FIAP dossiers, there are certain requirements for assessment of genotoxicity as follows.

“At least two in vitro assays should be performed. […] A positive result in genotoxicity testing would then require further assessment to determine whether it is genotoxic in vivo. […] One or more positive in vitro tests normally require follow-up by in vivo testing, unless it can be adequately demonstrated that the positive in vitro findings are not relevant for the in vivo situation. This is in line with the general strategy elaborated in the updated WHO/IPCS Harmonised Scheme on mutagenicity testing (Eastmond et al., 2009).” (page 16 - 17)

link to the FIAP guidance is http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2009.1305/epdf.

Thus the in vivo study was also preparation for approval according to the FIAP guidance. It in general takes years to develop a new enzyme and new markets. Registrant’s R&D and Market often seek various market opportunities in parallel. In addition many enzymes are already used for food manufacture as well as manufacture of non-food such as textile and paper. It was therefore a natural track for Registrant to follow FIAP guidance on this endpoint at early stages of R&D activities.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

The test material is a purified version of the batch PPW42035, as the test material was to be dosed intravenously to ensure maximum exposure of target tissue.

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Out-bred CD-1 mice (Crl:CD1(ICR))

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Obtained from Charles River (UK) Ltd., Margate, UK.
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 28-35 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Animals were housed in wire topped, solid bottomed cages, with (up to) three animals of the same sex per cage. Wood bedding (Aspen) along with wooden Aspen chew blocks and nesting material were provided.
- Diet: ad libitum access to 5LF2 EU Rodent Diet 14%
- Water: ad libitum via water bottles.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 15-20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light (0600 to 1800) and 12 hours dark

IN-LIFE DATES: From: 24 April 2017 To: 25 May 2017

Route of administration:

intravenous

Vehicle:

Phosphate buffered saline pH 7.4, with a composition of 10 mM KH2PO4/K2HPO4, 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2.

Details on exposure:

All treatments were given via intravenous bolus injection in order to maximise exposure of the target organ to the test article. Animals were dosed in ascending group order. The test article and vehicle control were given as two administrations, at 0 and
24 hours. This has been shown to be of sufficient duration for the expression of any genotoxic potential. The positive control was administered once only at 24 hours. All animals were sampled at 48 hours.

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Two administrations, at 0 and 24 hours.

Post exposure period:

24 hours

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 male and 12 female animals were dosed in the Range-Finder Experiment.
27 male animals were dosed in the Main Experiment.

Control animals:

yes

Positive control(s):

Cyclophosphamide (CPA) 40 mg/kg, single oral administration at 5 mL/kg on Day 2 (T=24 hours).

Tissues and cell types examined:

No tissues were retained from Range-Finder Experiment animals.
Both femurs were removed and bone marrow isolated from all Main Experiment animals at necropsy.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The maximum solubility of deoxyribonuclease, batch U8AGS in PBS is 53 mg/mL and the standard dose volume for bolus intravenous administration in mice is 5 to 10 mL/kg, therefore, the maximum feasible dose was 265 to 530 mg/kg/day. Based on this information an initial dose of 100 mg/kg/day was administered in a Range-Finder Experiment. Additional doses were tested until an estimate of the maximum tolerated dose (MTD) was determined. From the results of the Range-Finder Experiment dose levels of 100, 200 or 400 mg/kg/day 53 mg/mL Deoxyribonuclease, batch U8AGS, formulated in phosphate buffered saline (equivalent to 25% MTD, 50% MTD and the MTD respectively) were tested in the Main Experiment.

DETAILS OF SLIDE PREPARATION: Both femurs from each animal were exposed, removed, cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrows were flushed from the marrow cavity with 1 mL foetal bovine serum into appropriately labelled centrifuge tubes. A further 1 mL of foetal bovine serum was added to the tubes, and then centrifuged at 200 x 'g' for approximately five minutes; the serum was aspirated and a further 1 mL of foetal bovine serum was added and the centrifugation step was repeated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of two uniquely labelled slides. A smear was made from the drop by drawing the end of a clean slide along the labelled slide. Slides were air-dried and then fixed for 5 minutes in absolute methanol and rinsed several times in distilled water before being stained. One slide from each set was taken (the remaining slides kept in reserve). After rinsing several times in distilled water, slides were stained for 10 minutes in filtered Giemsa stain diluted in distilled water (typically 1:4 (v/v), actual ratio recorded in raw data). Slides were rinsed and dried before clearing in xylene for 3 minutes. When dry, slides were mounted with coverslips and stored at room temperature prior to analysis.

METHOD OF ANALYSIS: Scoring was carried out using light microscopy at an appropriate magnification. Slides from the vehicle and positive control animals were checked for quality and/or response prior to analysis. All slides were allocated a random code and analysed by an individual not connected with the dosing phase of the study. All animals per group were analysed. Initially the relative proportions of PCE, seen as purple/blue enucleate cells, and normochromatic erythrocytes (NCE), seen as pink/orange stained enucleate cells, were determined until a total of at least 500 cells (PCE plus NCE) had been analysed. Counting continued until at least 4000 PCE per animal where possible had been examined. All PCE containing MN observed during these two phases of counting were recorded.
The following criteria were used for analysis of slides:
1. Cells were of normal cell morphology
2. Areas where erythrocytes overlap were to be ignored
3. A MN was to be round or oval in shape
4. A cell containing more than one MN was scored as a single micronucleated cell
5. MN that were refractive, improperly stained or not in the focal plane of the cell were judged to be artefacts and were not scored.

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic / aneugenic damage if:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels.
2. The incidence and distribution of MN PCE exceeded the laboratory’s historical vehicle control data.
3. A dose-response trend in the proportion of MN PCE was observed.

The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met and bone marrow exposure was confirmed.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a dose-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between dose levels.

Statistics:

For each test article and vehicle control group, inter-individual variation in the numbers of MN PCE was estimated by means of a heterogeneity chi-square calculation (Lovell et al., 1989). The numbers of MN PCE in each treated group were compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine chi-square (Lovell et al., 1989). The tests were interpreted with one-sided risk for increased frequency with increasing dose. Probability values of p≤0.05 were accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 100, 265, 400, 530 mg/kg/day
- Solubility: Enzymes are water soluble
- Clinical signs of toxicity in test animals: 1/3 male animals dosed at 530 mg/kg/day died during the Day 3 observation period, confirming that 530 mg/kg/day exceeded the maximum tolerated dose for this study. A dose of 400 mg/kg/day produced moderate signs of toxicity and body weight loss but no morbidity or mortality in either male or female mice.
- Evidence of cytotoxicity in tissue analyzed: Not analysed
- Rationale for exposure: The maximum solubility of deoxyribonuclease, Batch U8AGS in PBS is 53 mg/mL and the standard dose volume for bolus intravenous administration in mice is 5 to 10 mL/kg, therefore, the maximum feasible dose was 265 to 530 mg/kg/day. The rodent bone marrow micronucleus test is recommended by various regulatory authorities as an appropriate test to determine the genotoxic potential of a compound in vivo.
- High dose with and without activation: 530 mg/kg/day

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Animals treated with all doses exhibited MN PCE frequencies that were similar to the concurrent vehicle control group.
- Ratio of PCE/NCE (for Micronucleus assay): Same as control
- Appropriateness of dose levels and route: 400 mg/kg/day identified as the MTD from the range-finding study. Intravenous dosing was applied to ensure maximum exposure of target tissue.
- Statistical evaluation: For each test article and vehicle control group, inter-individual variation in the numbers of MN PCE was estimated by means of a heterogeneity chi-square calculation (Lovell et al., 1989). The numbers of MN PCE in each treated group were compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine chi-square (Lovell et al., 1989). The tests were interpreted with one-sided risk for increased frequency with increasing dose. Probability values of p≤0.05 were accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.

Conclusions:

It was concluded that deoxyribonuclease, batch U8AGS, formulated in phosphate buffered saline did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male mice when dosed intravenously with up to 400 mg/kg/day (an estimate of the maximum tolerated dose for this study), under the experimental conditions employed.

Executive summary:

Deoxyribonuclease, batch U8AGS, formulated in phosphate buffered saline was tested for its potential to induce micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow of treated mice.

- Strain / Species: CD-1 mice.

- Vehicle: Phosphate buffered saline pH 7.4.

- Administration route: Intravenous bolus injection, to ensure systemic exposure.

- Dosing regimen: Two administrations at 0 hours (Day 1) and 24 hours (Day 2).

- Gender: Males only (no gender differences observed in the Range-Finder Experiment).

- Dose levels: 100, 200 or 400 mg/kg/day.

- Dose volume: 10 mL/kg.

- Maximum dose: Maximum tolerated dose based on Range-Finder data.

- Positive control: Cyclophosphamide (CPA) 40 mg/kg, single oral administration at 5 mL/kg on Day 2.

- Animals per group: Six (three for the positive control group).

- Clinical signs of toxicity: Following dosing on Day 2 and/or prior to necropsy on Day 3, observations of raised hair and/or hunched appearce were observed in the majority of animals dosed at 200 or 400 mg/kg/day. Body weight loss was observed in all animals dosed at 400 mg/kg/day (Day 1 to Day 3).

- Bone marrow sampled: 24 hours after the last dose administration (Day 3, equivalent to 48 hours).

- Assay validity: The vehicle control data (%PCE and MN PCE) were within the laboratory’s historical vehicle control data ranges. The positive control induced a significant increase in MN PCE that was comparable with the laboratory’s historical positive control data. The assay was therefore accepted as valid.

Animals treated with deoxyribonuclease, batch U8AGS, formulated in phosphate buffered saline at all doses exhibited group mean %PCE that were generally similar to the concurrent vehicle control group and which were within the laboratory’s historical vehicle control data, thus confirming there was no evidence of test article related bone marrow toxicity.

Animals treated with deoxyribonuclease, batch U8AGS, formulated in phosphate buffered saline at all doses exhibited MN PCE frequencies that were similar to the concurrent vehicle control group and that fell within laboratory's historical vehicle control data. There were no statistically significant increases in MN frequency for any of the groups receiving the test article, compared to the concurrent vehicle controls.

It was concluded that deoxyribonuclease, batch U8AGS, formulated in phosphate buffered saline did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male mice when dosed intravenously with up to 400 mg/kg/day (an estimate of the maximum tolerated dose for this study), under the experimental conditions employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the results obtained from the Ames test and in-vivo micronucleus study, deoxyribonuclease is not classified as having any genotoxic potential.