Pullulanase

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2012 - 11 September 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The purpose of the study was to satisfy regulatory demands because the enzyme is also used for production of food in EU.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

revised 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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

Constituent 2

Reference substance name:

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

Molecular formula:

Not available

IUPAC Name:

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

Constituent 3

Reference substance name:

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

Molecular formula:

Not available.

IUPAC Name:

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

Constituent 4

Reference substance name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Constituent 5

Reference substance name:

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

Molecular formula:

Not available.

IUPAC Name:

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

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Pullulanase, PPY34283
- Substance type: UVCB
- Physical state: liquid
- Lot/batch No.: PPY34283
- Expiration date of the lot/batch: Stable at least until 24 October 2022.
- pH: 5.6
- Stability under test conditions: The undiluted test material and 10% solutions in water are stable for at least 24 hours at room temperature or 5 degrees Celcius
- Storage condition of test material: minus 18 degrees of C

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, UK, Ltd
- Age at study initiation: 42 to 48 days
- Weight at study initiation: 219-276 g for males; 166-222 g for females
- Fasting period before study: None
- Housing: 5 of same sex per cage
- Diet : Standard pelleted rodent diet ad libitum
- Water : Tap water ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature : 19-23°C
- Humidity : 40-70 % RH
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2013-01-29 To: 2013-01-05

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 10, 33 and 100%, corresponding to 85, 281 and 851 mg total organic solids (TOS) per kg body weight per day.
- Amount of vehicle (if gavage): constant volume 10 mL/kg body weight.
- Purity: Water for formulation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose samples were analysed according to GLP.
Analysis of achieved concentration with regard to the enzyme activity was performed on samples taken once during weeks 1, 6 and 13. No significant differences were found between weeks and no significant differences were found between the achieved concentration for the high dose group and the 100% undiluted tox-batch, demonstrating satisfactory formulation.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

85 mg/kg bw/day (nominal)

Remarks:

1.0 mL/kg/day, equivalent to 0.085 g total organic solids (TOS)/kg/day nominal in water

Doses / concentrations 2

Dose / conc.:

281 mg/kg bw/day (nominal)

Remarks:

3.3 mL/kg/day, equivalent to 0.281 g total organic solids (TOS)/kg/day nominal in water

Doses / concentrations 3

Dose / conc.:

851 mg/kg bw/day (nominal)

Remarks:

10.0 mL/kg/day, equivalent to 0.851 g total organic solids (TOS)/kg/day nominal in water

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in this study were selected on the basis of results from studies performed on other similar enzyme preparations. The highest dose (10 mL/kg/day) was the maximum practical dose and represents administration of the enzyme, as received, at a volume-dose of 10 mL/kg body weight, which is the maximum practical volume-dose for repeat dose oral administration. The lower doses were selected using an approximate ratio of 3.3 between doses.

Positive control:

Not included

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Pre-dose and twice daily during the first week of treatment, twice daily during Weeks 2 to 5 and weekly thereafter, detailed observations were recorded

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each cage, i.e. sum of five animals: Yes weekly throughout the study

WATER CONSUMPTION: Yes
- Time schedule for examinations: Recorded by weight (over a 3 day period on each occasion) weekly throughout the study for each cage

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-treatment and during week 12
- Dose groups that were examined: Control and highest dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Isoflurane
- Animals fasted: yes, overnight
- How many animals: From all animals
- Parameters checked:
Haematocrit (Hct)
Haemoglobin (Hb)
Erythrocyte count (RBC)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Total white cell count (WBC)
Differential WBC count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)
Prothrombim time (PT)
Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13
- Animals fasted: yes, overnight
- How many animals: From all animals
- Parameters checked:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Total protein (Total Prot)
Albumin (Alb)
Albumin/globulin ratio (A/G Ratio)

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked:
Clarity and colour (App) - by visual assessment
Volume (Vol) - using a measuring cylinder
pH - using a pH meter
Specific gravity (SG) - by direct refractometry using a SG meter
Ketones (Keto)
Bilirubin/bile pigments (Bili)
Blood pigments (UBld)
Protein (Prot)
Creatinine (U-Creat)
Glucose (U-Gluc)


NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations: During week 12
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity: yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, full macroscopic examination
HISTOPATHOLOGY: Yes (Full list: Animals from Control and high dose group, Abnormalities only from mediate and low dose groups).

Other examinations:

FAECAL ANALYSIS: No

Weight of individual organs: Yes
- Time schedule for collection of organs: At necropsy

Statistics:

Statistical evaluation of grip strength, motor activity, bodyweight, haematology, blood chemistry, urinalysis, organ weights and any data derived from these was performed. The level of probability chosen as significant was p<0.05.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

All differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

All differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation

Urinalysis findings:

no effects observed

Description (incidence and severity):

It was considered that the high urinary volumes observed and low urinary pH may be due to a physiological adaptive response to the high salt content (> 4%).

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

HAEMATOLOGY:
All differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included increased reticulocyte and lymphocyte counts and small prolongations of activated partial thromboplastin times at all doses in females. For reticulocyte counts the differences from controls were small, none of the individual values was outside the background range (0.097x10^12 to 0.253x10^12/L; n=18) and there was no effect upon other erythrocyte parameters. The increased lymphocyte count at all doses in females was not dose-related and none of the individual values was abnormal. In addition, the group mean value for the controls (3.35x10^9/L) was well below the mean of the background data (6.85x10^9/L; n=307) and this finding was therefore a consequence of lower than expected values in the control group. The small prolongation of activated partial thromboplastin time similarly lacked dose relationship and all values were well within the background range (11.1 to 21.2 seconds; n=308). Consequently, each of these trends was considered fortuitous.

CLINICAL CHEMISTRY
All differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the low creatinine concentrations in males receiving 100% Pullulanase, PPY34283, since the group mean value of the control group was raised by one unusually high individual value (No. 6: 76 μmol/L) and all individual values in the high dose group were within the background range (90 percentile range 27 to 46 μmol/L; n=299). They also included the low potassium concentrations in males receiving 100% Pullulanase, PPY34283, where all values were within the background range (90-percentile range 4.0 to 6.1 mmol/L; n=289).

URINALYSIS:
The urinalysis investigation in Week 13 revealed, when compared with the controls, high urinary volumes associated with slightly low specific gravity at all doses in males, but particularly those receiving 33 or 100% Pullulanase, PPY34283 where statistical significance was attained. In males and females given 100% Pullulanase, PPY34283 there was a clear reduction of urinary pH. It was considered that this may be due to a physiological adaptive response to the high salt content of the Toxbatch (> 4%).

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 851 mg/kg bw/day (nominal)

Based on:

other: total organic solids (TOS)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) in rats was 10 mL of the undiluted pullulanase/kg body weight/day equivalent to 851 mg Total Organic Solids (TOS)/kg body weight/day.

Executive summary:

The repeated dose oral toxicity study was a subchronic toxicity test conducted according to OECD guideline 408 (revised 1998), and in compliance with GLP. The purpose of the study was to satisfy regulatory demands because the enzyme is used for production of food in EU.

In conclusion, oral administration (by gavage) of pullulanase to rats at dosages of up to the highest dose level tested, equivalent to 10 mL of the undiluted test material/kg body weight/day or 851 mg Total Organic Solids (TOS)/kg body weight/day for thirteen weeks was well-tolerated and did not produce any toxicologically significant changes.

Consequently, the No Observed Adverse Effect Level (NOAEL) was considered to be the highest dose level administered, equivalent to 10 mL of the undiluted pullulanase/kg body weight/day or 851 mg Total Organic Solids (TOS)/kg body weight/day.