Pullulanase

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

October 26, 1993-August 24, 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

The OECD guideline came later (in 1997) but the overall principles of the guideline was followed in the study.
Single treatment (IP) performed, followed by 12, 24 and 48 hour sampling of bone marrow.

GLP compliance:

yes

Remarks:

incl. certificate

Type of assay:

other: micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Pullulanase

EC Number:

232-983-9

EC Name:

Pullulanase

Cas Number:

9075-68-7

Molecular formula:

not available

IUPAC Name:

Pullulanase

Test material form:

solid: particulate/powder

Details on test material:

- Substance type: UVCB
- Physical state: Powder
- Lot/batch No.: Not provided in report
- Expiration date of the lot/batch: not provided in report
- Stability under test conditions: The test substance and dilutions in water are stable for at least 24 hours at room temperature or 5 degrees Celcius
- Storage condition of test material: At room temperature with desiccant

Test animals

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

Weight: 23 to 28g
From: Charles River Laboratories, Portage, Michigan
Randomized: removed randomly from shipping cages and randomly assigned to cages
Cages: microisolator
No. of mice/cage: 5 (sex separated)
Food: Purina Rodent Laboratory Chow No. 5002C ad libitum
Water: ad libitum
Acclimatization: 6 days before dosing
Mice identification: tail mark
Room tempreature: 19-22 degrees C
Humidity: 24-78%

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Phosphate buffered saline (PBS)

Details on exposure:

10 mL/kg
IP to 15 males and 15 females/ Pullulanase/ dose; 5 doses in total
IP to 5 males and 5 females / positive control
IP to 5 males and 5 females / vehicle control

Duration of treatment / exposure:

Pullulanase injected mice were killed 12, 24 and 48 hours after dosing
Positive- and vehicle control mice were killed 24 hours after dosing

Frequency of treatment:

Single injection

Post exposure period:

Mice were killed 12, 24 and 48 hours after dosing.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

889 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 581 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

2 811 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females/dose/time point = 15/sex/dose

Control animals:

yes

Positive control(s):

0.4 mg triethylenemelamine (TEM)/kg b.w., dosed by ip injection.
TEM and the dose 0.4 mg/kg was selected because this was known to give reproducible increase in the frequency of micronucleated PCEs in Swiss Webster mice.

Examinations

Tissues and cell types examined:

bone marrow from both femora

Details of tissue and slide preparation:

One 15-ml centrifuge tube containing 750 uL of bovine calf serum was prepared for each animal.
Both femora from mice killed by cervical dislocation were cleaned, and the bone marrow canal flushed with 1 mL disposable syringe with 25 G needle with 0.2 mL fetal calf serum into the centrifuge tube. Cells were centrifuged for 5 min. at 100 x g, the supernatant was decanted, pellet was mixed in the remaining fluid, and the suspended cells were placed onto slides. The drop was spread by spreader slide to even film, slide was air dried, fixed in methanol for 2 minutes, and dried.

Two slides were prepared per animal. Slides were stained for 20 min. in 5% Giemsa (in PBS with 3% methanol and 3% 0.1M citric acid), rinsed in DI water, and air dried. Slides were protected by coverslip attached with Permount, and analyzed at 100X, oil immersion, magnification.

Preliminary evaluation with uncoded slides (dose range finding):
% of PCEs (polychromatic erythrocytes) in 1000 erythrocytes from one animal per sex, from each top dose and vehicle control at 12, 24 and 48 hours after exposure were evaluated to select the highest dose. No clear depression of PCEs was detected, therefore the highest dose was selected as the highest dose scored.

Evaluation with coded slides:
Micronuclei were scored from male and female slides at 12, 24 and 48 hours after exposure to the highest dose, the next two lower doses, positive and negative controls. Slides were divided into two identical groups and coded by individual not involved in scoring. Two observers were utilized, one for each set of slides.
When slides were decoded and tabulated, the ratios of PCEs per 5000 erythocytes, and the number of micronucleated cells per 1000 PCEs and per 1000 NCEs were calculated per each treatment group.

Evaluation criteria:

Micronuclei are round and oval shaped bodies found in the cytoplasm; refractile and improperly shaped cells are not scored. Cells with more micronuclei are scored as a single micronucleated cell.

Criteria for interpretation:
Positive:
There is dose-related increase in micronuclei at at least one collection time.
And if one or more doses induces a statistically significant (p<0.05) increase in micronuclei induction.

Negative:
The positive criteria are not met.

Both biological and statistical significance are considered together.

Statistics:

No statistical analysis was required to evaluate results.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
Pullulanase did not induce micronuclei (clastogenic effects) in male or female mice at doses up to 5000 mg Pullulanase/kg bw when injected by IP injection.

Executive summary:

In a dose range-finding study 3 male and 3 female mice/dose received vehicle (PBS 10 mL/kg), 50, 158.1, 500, 1581, or 5000 mg (half-log doses) Pullulanase/kg by ip injection. The top dose 5000mg/kg was not toxic to mice and selected as the highest dose for the micronucleus testing.

In the main study, the micronuclei from bone marrow of 15 male and 15 female mice/dose were analyzed. Mice were given one of 5 doses, 500, 889, 1581, 2811 or 5000 mg Pullulanase/kg, by ip injection. Five male and 5 female mice were exposed to vehicle or positive control. Bone marrow cells were collected from 5 male and female mice/time killed 12, 24 and 48 hours after exposure. Cells were collected by flushing the bone marrow canal of the femur with fetal bovine serum. Cells were centrifuged, smeared on slides, dried, fixed with methanol, stained with Giemsa and analyzed at 100x (oil immersion) magnification. Analyzed were polychromatic erythrocytes (PCEs) per 1000 erythrocytes.

The positive control, 0.4 mg triethylenemelamine (TEM), induced some toxicity, as evidenced by depression of percentage of newly-formed PCEs, and TEM elevated the number of micronuclei in PCEs from male and female mice. No toxic effects were observed in the bone marrow cells and no significant number of micronuceli were induced in mice of either sex, exposed to 1581, 2811 or 5000 mg Pullulanase/kg. Pullulanase did not induce micronuceli (clastogenic effects) in male or female mice.