Registration Dossier

Administrative data

Description of key information

It was concluded that oral administration of beta-amylase, batch PPY36295 to Sprague-Dawley rats at doses up to 100% of the beta-amylase batch (equivalent to 1.199 g TOS/kg/day) for 90 days was well-tolerated and did not cause any adverse change. The no-observed adverse-effect level (NOAEL) was considered to be 100% of the beta-amylase batch, equivalent to 1.199 g TOS/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-07-2014 to 15-12-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 42-48 days
- Weight at study initiation: 217 to 274 g for males; 162-218 g for females
- Fasting period before study: None
- Housing: 5 animals of the same sex per cage
- Diet: Teklad 2014C Diet ad libitum (removed overnight before blood sampling for hematology or blood chemistry).
- Water: Ad libitum. Potable water from the public supply via polycarbonate bottles with sipper tubes.
- 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: 22 April 2014 To: 12 September 2014
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
reverse osmosis water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test enzyme was provided deep-frozen in a number of containers. The day before formulation, the frozen aliquots for that day were removed from the freezer and allowed to thaw overnight in a refrigerator (approximately 4°C).
VEHICLE
- Concentration in vehicle: 10, 33 and 100%, corresponding to 0.120, 0.396 and 1.199 g TOS/kg/day
- Amount of vehicle (if gavage): constant volume 10 mL/kg body weight.
- Purity: Reverse osmosis water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose samples were analysed according to GLP.
Samples of each formulation prepared for administration in Weeks 1, 6 and 13 of treatment were analysed for achieved concentration of the test substance. A total of 6 x 5 mL samples were taken from the middle of each formulation (Groups 1 to 4) that was prepared for administration in Weeks 1, 6 and 13, and all samples were frozen at approximately -20°C upon completion of sampling. Three samples from each formulation/occasion were subsequently dispatched (deep frozen on dry ice) to Novozymes A/S responsible for formulation analysis.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Dose / conc.:
0.12 other: g TOS/kg/day
Dose / conc.:
0.396 other: g TOS/kg/day
Dose / conc.:
1.199 other: g TOS/kg/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The test enzyme was not anticipated to cause any significant findings, based on the results obtained from studies with similar materials. The highest dose (100%) is the maximum practical dose and represents administration of the enzyme, as received, at a volume dosage of 10 mL/kg body weight. The lower doses were selected using a ratio of approximately 3.3 between doses.
Positive control:
Not included
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cages were inspected daily for evidence of animal ill-health amongst the occupants.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced (Week -1), on the day that treatment commenced (Week 0), weekly throughout the treatment period and before necropsy.

FOOD CONSUMPTION:
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started (Week -1) and for each week throughout the treatment period.

WATER CONSUMPTION:
- Time schedule for examinations: Fluid intake was assessed by daily visual observation.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment started and during week 12.

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 leucocyte count (WBC), Platelet count (Plt), Prothrombim time (PT), Activated partial thromboplastin time (APTT)

Differential WBC count:
Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)

Morphology:
Anisocytosis, Microcytosis, Macrocytosis, Hypochromasia, Hyperchromasia

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), 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: The macroscopic examination performed after 90 days of treatment.

HISTOPATHOLOGY: The macroscopic examination performed after 90 days of treatment.
Other examinations:
Weight of individual organs: Yes
- Time schedule for collection of organs: At necropsy
Statistics:
The main tests used were Barlett's test, Dunnett’s test, Shirley’s test, Williams’ test, Fisher’s Exact test, analysis of covariance. Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significant increase of overall weight gain at all doses in males, where the weight gains were approximately 14-20% higher than the controls, was considered of no toxicological importance since there was no dose response and no similar trend in females.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was slightly higher than that of the controls from Week 2 at all doses in males, reflecting the higher bodyweight gain reported for these animals. There was, however, no dose response as the average food consumption of the males receiving 100% of the beta-amylase batch was slightly lower than that of the low and intermediate dose groups. Moreover, there was no similar finding in females and, consequently, this was considered of no toxicological importance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Platelet counts were decreased at all doses in females, with the magnitude of the difference from controls being greatest in those receiving 100% of the beta-amylase batch. The majority of individual values, including the controls, were below the 90 percentile background range but when compared with concurrent controls, 6/10 high dose animals, 2/10 intermediate dose and 2/10 low dose animals had individual values below the control range. In the absence of any change of clotting times (prothrombin and activated partial thromboplastin time) this reduction of platelet count was considered of no toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
High plasma potassium concentrations were observed at all doses in males, however, there was no dose-response, no similar finding in females and, with the exception of only one animal in each treated group, all individual values were within the background range.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The group mean forelimb grip strength for females receiving 33 or 100% of the Beta-amylase batch was slightly but statistically significantly higher than that of the controls. All group mean scores were, however, within the historical control data range and there was no similar trend in the males. These inter-group differences were therefore considered of no biological significance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 199 other: mg TOS/kg/day
Based on:
other: total organic solids (TOS)
Sex:
male/female
Basis for effect level:
other: No adverse effects were seen so NOAEL was the highest dose administered.
Critical effects observed:
no
Conclusions:
It was concluded that oral administration of beta-amylase, batch PPY36295 to Sprague-Dawley rats at doses up to 100% of the beta-amylase batch (equivalent to 1.199 g TOS/kg/day) for 90 days was well-tolerated and did not cause any adverse change. The no-observed adverse-effect level (NOAEL) was considered to be 100% of the beta-amylase batch, equivalent to 1.199 g TOS/kg/day.
Executive summary:

The objective of this study was to assess the systemic toxic potential of beta-amylase batch PPY36295 when administered orally by gavage to Sprague-Dawley (Crl:CD(SD)) rats for 90 days. Three groups, each comprising 10 males and 10 females, received doses of 10, 33 or 100% of the beta-amylase batch (equivalent to 0.120, 0.396 or 1.199 g TOS/kg/day). A similarly constituted control group received the vehicle (reverse osmosis water) at the same volume-dose (10 mL/kg body weight).

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (by visual assessment), ophthalmic examination, haematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

General appearance and behaviour, sensory reactivity responses, grip strength and motor activity were not affected by treatment, there were no deaths during the treatment period and there was no effect of treatment on bodyweight gain or on food and water consumption. There were no treatment-related ophthalmic findings. The haematology and blood chemistry investigations during Week 13 did not identify any toxicologically significant differences from controls. Organ weights were unaffected and there were no treatment-related macroscopic or microscopic findings.

It was concluded that oral administration of beta-amylase, batch PPY36295 to Sprague-Dawley rats at doses up to 100% of the beta-amylase batch (equivalent to 1.199 g TOS/kg/day) for 90 days was well-tolerated and did not cause any adverse change. The no-observed adverse-effect level (NOAEL) was considered to be 100% of the beta-amylase batch, equivalent to 1.199 g TOS/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 199 mg/kg bw/day
Quality of whole database:
Toxicological data has been generated within the enzyme producing industry during the last 40 years. Substantial documentation on the safety of the production strains has been generated, and the enzyme test materials are thoroughly characterized. High quality studies for all relevant endpoints for in vivo studies as well as in vitro studies show that industrial enzymes from well-known and well characterized production strains have very similar safety profiles across the catalytic activities. Read-across can therefore be applied for the majority of toxicological endpoints. The database can therefore be considered of high quality.

Additional information

Justification for classification or non-classification