Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01-12-2016 to 16-08-2017, (study protocol amended on 01-08-2017)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
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:
liquid
Details on test material:
- Lot/batch No.: PPT42347
- Expiration date of the lot/batch: October 2026
- Stability under test conditions: Stable for at least 168 hours.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
RccHan™:WIST rat.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Limited
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 41-47 days
- Weight at study initiation: 121 to 173 g for males; 113-148 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: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature : 20-24°C
- Humidity : 40-70 % RH
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 January 2016 To: 18 April 2016

Administration / exposure

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 use, the required numbers of test material containers were transferred from the freezer to nominally 4ºC storage to allow them to thaw. On the day of formulation the test material containers were inverted 10 times and gently magnetically stirred. The required amount of test material was measured out and the residue returned to the freezer as quickly as practicable. The required volume of vehicle was added to the pre-measured material and gently mixed by magnetic stirring and inversion to avoid any potential damage to the protein and ensure homogeneity of the dose preparation. Formulations were retained refrigerated (2-8°C) overnight (when prepared the day before) or at ambient temperature.
VEHICLE
- Concentration in vehicle: 10, 33 and 100%, corresponding to 112, 368 and 1116 mg total organic solids (TOS) /kg bw/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.
Stability of formulations at 10 and 100%, when retained for up to 24 hours at refrigerated (nominally 5°C) or ambient temperature was demonstrated b y Novozymes A/S. 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 10 mL samples were taken from the middle of each formulation 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 the Principal Investigator responsible for formulation analysis. All remaining samples were retained (at approximately -20°C) as a contingency but were discarded after finalization of the formulation analysis report since no further analysis was required.

Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
131 mg/kg bw/day (actual dose received)
Remarks:
Total organic substance
Dose / conc.:
433 mg/kg bw/day (actual dose received)
Remarks:
Total organic substance
Dose / conc.:
1 312 mg/kg bw/day (actual dose received)
Remarks:
Total organic substance
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

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily

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, on the day that treatment commenced (Week 0), weekly throughout the study 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 and for each week throughout the treatment period.

WATER CONSUMPTION:
- 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: All animals before treatment. Control and highest dose group after treatment.

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)
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)

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

HISTOPATHOLOGY: Yes
Other examinations:
FAECAL ANALYSIS: No

Hematology, Bone Marrow: Yes

Weight of individual organs: Yes
- Time schedule for collection of organs: At necropsy
Statistics:
The main tests used were Dunnett’s test, Shirley’s test, Williams’ test. Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One incidental and non-treatment related death occurred in this study. One Control female was killed for welfare reasons in Week 9 after displaying decreased activity, laboured breathing, gasping, distended abdomen and piloerect coat. The macroscopic examination indicated that the cause of the respiratory distress and abdominal distension to be the presence of gaseous distension of the stomach and intestinal tract, though the histopathological examination suggested that the condition of the animal was due to lesions reported in the kidneys (moderate pelvic dilation).
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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):
All inter-group differences from control, including those attaining statistical significance, were minor, lacked dose relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the small but statistically significant decrease of mean cell hemoglobin concentration in males given 100% trehalase but since the extent of the difference from controls was small and at this dose only a single animal had a marginally lower value than concurrent control and all individual values were within the background range it was attributed to normal biological variation.
In males given 33 or 100%, a statistically significant increase in monocyte counts, but this was attributable mainly to a low group mean value for controls caused by the individual values of 5/10 animals being below the background range, with almost all individual values for males at 33% (9/10 animals) or 100% (8/10 animals) trehalase being within the background range and, furthermore, the individual values that were outside the background range were within the concurrent control range.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
All inter-group differences from control, including those attaining statistical significance, were minor, lacked dose-relationship or were confined to one sex and could all, therefore, be attributed to normal biological variation. Slight but statistically significant increases of plasma creatinine occurred at all doses in males, and also in females receiving 100% trehalase, batch PPT42347, but the extent of the differences from controls was small in each case and all individual values were within the background range.
There was also a slight but statistically significant decrease of potassium concentrations in females given 100% trehalase, with the majority of animals having values that were just below the background range, however those values were generally within the concurrent control range (there were many individual values for females across the groups, including controls, that had plasma potassium concentrations that were also below the background range). Consequently, and also in the absence of any other findings that were indicative of an effect on kidney function in this study, these differences were attributed to normal biological variation and were therefore not related to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
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

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 312 other: mg TOS/kg bw/day
Based on:
other: total organic solids (TOS)
Sex:
male/female
Basis for effect level:
other: Trehalase was well-tolerated and did not cause any adverse change.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
It is concluded that oral administration of trehalase, batch PPT42347 to Han Wistar rats at doses up to 100% of trehalase, batch PPT42347 for 13 weeks was well-tolerated and did not cause any adverse change. The no-observed adverse-effect level (NOAEL) was therefore considered to be 100% of the trehalase, batch PPT42347 (equivalent to 1312 mg TOS/kg body weight/day).
Executive summary:

The objective of this study was to assess the systemic toxic potential of trehalase, batch PPT42347 when administered orally by gavage to Han Wistar rats for 13 weeks. Three groups, each comprising ten males and ten females, received doses of 10, 33 or 100% of Trehalase, batch PPT42347 (equivalent to 131, 433 and 1312 mg TOS/kg bw/day). A similarly constituted control group received the vehicle (reverse osmosis water) at the same volume-dose (10 mL/kg body weight) as the treated groups.

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

There were no deaths associated with trehalase, batch PPT42347. The general appearance and behavior, sensory reactivity responses, grip strength and motor activity were not affected by treatment during the study. A control female was killed for welfare reasons in Week 9, but this was clearly an incidental death. There was no effect of treatment on body weight, food consumption or (visually assessed) water consumption. There were no treatment-related ophthalmic findings. The hematology and blood chemistry investigations during Week 13 did not identify any treatment-related findings. Organ weights were unaffected by treatment and there were no treatment-related macroscopic or histopathological findings.

It is concluded that oral administration of trehalase, batch PPT42347 to Han Wistar rats at doses up to 100% of trehalase, batch PPT42347 for 13 weeks was well-tolerated and did not cause any adverse change. The no-observed adverse-effect level (NOAEL) was therefore considered to be 100% of the trehalase, batch PPT42347 (equivalent to 1312 mg TOS/kg body weight/day).