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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 June 2009 - 20 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
revised 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): Amylase AZ, PPY 29933
- Substance type: UVCB
- Physical state: liquid
- Lot/batch No.: PPY 29933
- Expiration date of the lot/batch: Stable at least until 13 May 2019.
- pH: 7.4
- Stability under test conditions: The undiluted test material and 10% solutions in water are stable for at least 24 hours at room temperature or 4 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 and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK, Ltd
- Age at study initiation: 43 days
- Weight at study initiation: 176-221 g for males; 150-189 g for females
- Fasting period before study: None
- Housing: 5 animals per cage, separate sex
- Diet : Standard pelleted rodent diet ad libitum
- Water : Tap water ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature : 19-23°C (on one occasion during week 13 of treatment a minimum temperature of 17°C was recorded but this minor deviation from target was considered to have no effect on the health and welfare of the animals and no effect on the results of the study)
- Humidity : 40-70 % RH
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2009-07-01 To: 2009-10-09

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 110, 362 and 1100 mg total organic solids (TOS) per kg body weight.
- 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 / concentrationsopen allclose all
Dose / conc.:
1 other: mL/kg bw/day
Remarks:
corresponding to 0.110 g TOS/kg/day
Dose / conc.:
3.3 other: mL/kg bw/day
Remarks:
corresponding to 0.362 g TOS/kg bw/day
Dose / conc.:
10 other: mL/kg bw/day
Remarks:
corresponding to 1.10 g TOS/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 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: 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: Daily by visual observation

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment started 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: Yes (isoflurane anaesthesia)
- 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: No


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. Adrenals, brain, femur with joint, heart, kidneys, liver, lungs, spinal cord, sternum, stomach, thyroid and uterus were preserved and for the control group and the high dose group these tissues were examined.
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
Description (incidence and severity):
One death of a high dose female was clearly accidental.
Mortality:
no mortality observed
Description (incidence):
One death of a high dose female was clearly accidental.
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):
Small increase of lymphocyte numbers at high dose in both sexes. No histopath evidence of inflammatory changes in any tissue. Compared to background data for lymphocyte counts the study values were not abnormal. Considered of no toxicological significance
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Small increase of plasma potassium concentration in high dose males. No evidence of any effect upon the kidneys or water balance and no similar finding in females. The change was therefore considered of no toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Slightly lower spleen weight in high dose females. Males not affected. No histopathological evidence of inflammatory changes in any tissue and no evidence of immunosuppression. The finding was therefore considered to be of no toxicological significance.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
HAEMATOLOGY AND ORGAN WEIGHTS: A small increase of lymphocyte numbers in the peripheral blood was reported at high dose (1100 mg TOS/kg body weight/day) in both sexes, but particularly for the females in which there was also a small reduction of spleen weight. There was no evidence at the histopathological examination for any chronic inflammatory change in any tissue and immunosuppression was discounted because this would result in low (not high) lymphocyte counts. Review of the findings against the background control data for lymphocyte counts also demonstrated that the study values were not abnormal. These findings were, therefore, considered not to be of any toxicological significance and there was no evidence that alpha-amylase, PPY 29933 had any adverse effect on the immune system.


Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 100 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

Key result
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 alpha-amylase/kg body weight/day equivalent to 1100 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 alpha-amylase 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 1100 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 alpha-amylase /kg body weight/day or 1100 mg Total Organic Solids (TOS)/kg body weight/day.