Amylase, β-

Endpoint summary

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 1324 mg enzyme concentrate dry matter/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 1324 mg enzyme concentrate dry matter/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

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.

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

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD)

Sex:

male/female

Details on test animals or test system 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 132.5, 437 and 1324 mg enzyme concentrate dry matter/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.:

132.5 other: enzyme concentrate dry matter/kg/day

Dose / conc.:

437 other: enzyme concentrate dry matter/kg/day

Dose / conc.:

1 324 other: enzyme concentrate dry matter/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:

1 324 mg/kg bw/day (actual dose received)

Based on:

other: enzyme concentrate dry matter

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 1324 mg enzyme concentrate dry matter/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 1324 mg enzyme concentrate dry matter/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 132.5, 437 and 1324 mg enzyme concentrate dry matter/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 1324 mg enzyme concentrate dry matter/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 1324 mg enzyme concentrate dry matter/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 324 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.