Amylase, gluco-

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 November 2005 - 5 October 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This assessment of sub-chronic systemic toxicity (according to OECD TG 408) has been performed due to data requirements from the European Food Safety Authority (EFSA), as this enzyme also comply with the regulatory system of FIAP [REGULATION (EC) No 1331/2008 and EFSA CEF guidance from 2009/2013]. Moreover; it has been generated in accordance with Directive 2010/63/EU.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of glucan 1,4-alpha-glucosidase (EC no. 232-877-2, CAS no. 9032-08-0, EC name: Amylase, gluco-, Enzyme Class No.: 3.2.1.3)

Molecular formula:

Not available.

IUPAC Name:

Active enzyme protein of glucan 1,4-alpha-glucosidase (EC no. 232-877-2, CAS no. 9032-08-0, EC name: Amylase, gluco-, Enzyme Class No.: 3.2.1.3)

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:

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

Molecular formula:

Not available. See remarks.

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. See remarks.

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. See remarks.

IUPAC Name:

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

Test material form:

liquid

Details on test material:

- Lot/batch No.: PPY24900
- Expiration date of the lot/batch: 10 May 2015

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: 45-49 days
- Weight at study initiation: 230-293 g for males; 150-191 g for females
- Fasting period before study: None
- Housing: 5 animals per cage, separate sex
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 12 days

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

IN-LIFE DATES: From: 2005-11-16 To: 2006-02-28

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Purified water obtained by reverse osmosis

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 10, 33 and 100%, corresponding to 15, 50 and 152 mg enzyme concentrate dry matter/mL
- Amount of vehicle (if gavage): constant volume 10 mL/kg b.w.
- Purity: Purified water obtained by reverse osmosis

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of achieved concentration with regard to the enzyme activity was performed on samples taken once during weeks 1, 6 and 13. The mean achieved enzyme activities were between 98.7 to 102% of the intended, demonstrating satisfactory formulation.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 519.2 mg/kg bw/day (nominal)

Remarks:

Expressed in mg enzyme concentrate dry matter/kg bw/day

Doses / concentrations 2

Dose / conc.:

501.3 mg/kg bw/day (nominal)

Remarks:

Expressed in mg enzyme concentrate dry matter/kg bw/day

Doses / concentrations 3

Dose / conc.:

151.9 mg/kg bw/day (nominal)

Remarks:

Expressed in mg enzyme concentrate dry matter/kg bw/day

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 bodyweight, 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 twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during the first week of treatment, twice weekly during Weeks 2 to 4 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, was determined and mean daily diet consumption per group calculated as g feed/rat/week: Yes

FOOD EFFICIENCY:
- Body weight gain in g/food consumption in g per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION : Yes
- Time schedule for examinations: weekly, over a 3-day period in each week

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment started and during week 13
- 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: Yes (isoflurane anaesthesia)
- Animals fasted: yes, overnight
- How many animals: From all animals
- Parameters checked:
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)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13
- Animals fasted: yes, overnight
- How many animals: From all animals
- Parameters checked:
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyl transpeptidase (gGT)
Total bilirubin (Bili)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

URINALYSIS: No

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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

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, 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

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

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:

effects observed, treatment-related

Description (incidence and severity):

The changes that were identified were not considered of toxicological significance.

Histopathological findings: neoplastic:

not specified

Details on results:

HISTOPATHOLOGY: NON-NEOPLASTIC : Histopathology indicated an increased incidence of cortical vacuolisation in the adrenal glands of males in the high and mid dosed groups. This was considered a slight exacerbation of a normal background finding in young CD rats, which is not toxicologically significant.
At the time of the study, the issue of cortical vacuolation was addressed by expanding the microscopic examination of the adrenals to include group 2 and 3, in addition to group 1 (control) and group 4 (high dose group), to have the full dose-response pattern of the cortical vacuolation seen in the adrenal glands of males (pls see table for results in next information box). Further, the negative control rats from seven unrelated and recent 13-week oral toxicity studies were investigated to get useful baseline information on this specific parameter for the used strain of rats.

The reason why the small increase in the incidence and severity of cortical vacuolation in the adrenal gland of males is regarded of no toxicological significance is based on the following elements:

1. The cortical vacuolation in the adrenals was the only effect seen, there was no indication of degeneration, inflammation or necrosis, and the females did not show this effect at all. Further, the organ weight of the adrenals was not increased. This is important and indicates that the homeostasis of the adrenals was fully retained. The cortical vacuolation in the males was therefore not regarded as a toxicological response but rather a normal physiological response of the adrenal cortex (the secretion of glucocorticoids) in response to the increasing dose of protein as a result of the administration of the enzyme.
Minimal vacuolation of the adrenal cortex was also reported in two untreated Control males of the present study, i.e. incidence 20%, and in up to 42% of untreated animals from the other recent 13-week studies. Therefore, the result of the present study lies within the normal range of this rat strain.

2. The Contract Research Organisation, Huntingdon Life Sciences, Ltd., Cambrigdeshire, England, is a top quality institute with long term experience in preclinical testing with lots of experts in toxicology and pathology. The present conclusion is based on their evaluation, which adds to the solidity of the present data and conclusion.

It is therefore concluded that the findings in the adrenals of the male rats are unlikely to be of any toxicological significance.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 519.2 mg/kg bw/day (nominal)

Based on:

other: Expressed in mg enzyme concentrate dry matter/kg bw/day

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The cortical vacuolation was found in the adrenals of the males only (for further background and discussion, please see details on results of histopathology above) :

 

   

Group/sex			1M	2M	3M	4M
Dose (mL/kg/day)			0	1.0	3.3	10.0
Cortical vacuolation		Total	2	2	4	5
		Minimal	2	2	3	4
		Slight	0	0	1	1
Number of animals examined			10	10	10	10

 

 

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) in rats is 10 mL of the undiluted glucoamylase batch/kg bw/day equivalent to 1519.3 mg enzyme concentrate dry matter/kg bw/day.

Executive summary:

The repeated dose oral toxicity was a subchronic toxicity test conducted according to OECD guideline 408 (adopted 1998), and in compliance with GLP. 

In conclusion, oral administration (by gavage) of glucoamylase, PPY 24900, to rats at dosages of up tothe highest dose level tested, equivalent to 10 mL of the undiluted Glucoamylase batch/kg bw/day or 1519.3 mg enzyme concentrate dry matter/kg bw/day for thirteen weeks was well-tolerated and did not produce any toxicologically significant changes. Necropsy and the following microscopic examination revealed no treatment related effects apart from an increased incidence or increased severity of cortical vacuolation in the adrenal glands of males given 3.3 or 10.0 mL/kg/day, but this was considered a slight exacerbation of a normal background finding in young CD rats which was not considered toxicologically significant.

Consequently, the No Observed Adverse Effect Level (NOAEL) was considered to be the highest dose level administered, equivalent to 10 mL tox batch/kg bw/day or 1519.3 mg enzyme concentrate dry matter/kg bw/day.