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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 November 2018 - 29 August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Active enzyme protein of Subtilisin (EC no. 232-752-2, CAS no. 9014-01-1, EC name: Subtilisin, Enzyme Class No.: 3.4.21.62)
Molecular formula:
Not avaiable
IUPAC Name:
Active enzyme protein of Subtilisin (EC no. 232-752-2, CAS no. 9014-01-1, EC name: Subtilisin, Enzyme Class No.: 3.4.21.62)
Constituent 2
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 3
Reference substance name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available.
IUPAC Name:
Lipids 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.
IUPAC Name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
Constituent 5
Reference substance name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Test material form:
liquid
Details on test material:
- Lot/batch No.: PPA55402
- Expiration date of the lot/batch: 22 June 2028

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Limited.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 43 to 49 days
- Weight at study initiation: Males: 133-183 g, Females: 120-157 G
- Fasting period before study: None
- Housing: Polycarbonate body with a stainless steel mesh lid, changed at
appropriate intervals.
- Diet (e.g. ad libitum): Teklad 2014C Diet ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%
- Air supply: Filtered fresh air which was passed to atmosphere and not
recirculated.
- Photoperiod: 12 hrs light/12 hrs dark

IN-LIFE DATES: From: 5 December 2018 To: 7 March 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Reverse osmosis water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 57.2, 188.8 and 572.0 mg enzyme concentrate dry matter/kg bodyweight
- Amount of vehicle (if gavage): 5 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
57.2 mg/kg bw/day (nominal)
Remarks:
enzyme concentrate dry matter
Dose / conc.:
188.8 mg/kg bw/day (nominal)
Remarks:
enzyme concentrate dry matter
Dose / conc.:
572 mg/kg bw/day (nominal)
Remarks:
enzyme concentrate dry matter
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Fasting period before blood sampling for clinical biochemistry: Food removed overnight
Positive control:
No

Examinations

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: One week before treatment, on the day that the treatment commenced, once a week throughout the study and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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 study.

WATER CONSUMPTION: Fluid intake was assessed by daily visual observation. No significant effect was observed and, consequently, quantitative measurements were not performed

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretreatment and week 12
- Dose groups that were examined: all animals at pretreatment and control group and highest dose group at week 12.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked:
Hematocrit (Hct), Hemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell hemoglobin (MCH)*, Mean cell hemoglobin concentration (MCHC)*, Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC)

Differential leucocyte 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: all animals
- Parameters checked:
Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Bile acids (Bi Ac), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Cholesterol (HDL), Cholesterol (LDL), sodium (Na), Potassium (K), Chloride (Cl), Total protein (Total Prot), Albumin (Alb)

PLASMA/SERUM HORMONES: Yes
- Time of blood sample collection: At termination
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked:
Triiodothyronine (T3), Thyroxine (T4), Thyroid stimulating hormone (TSH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 12
- Dose groups that were examined: All animals
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER:
Estrus cycles: Wet smears were taken from the vagina of all females using pipette lavage for 4 days before scheduled necropsy. Smears were assessed to establish the stage of estrus (metestrus, diestrus, proestrus and estrus) and were used to assist in the histological evaluation of estrogen sensitive tissues.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
A male animal in the highest dose group died in week 11. Lung lesions were considered the major contributing factor to death and were most likely caused by reflux and/or aspiration of dose. Consequently, this death was attributed to the dose administration procedure and not to toxicity.
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:
no effects observed
Clinical biochemistry findings:
no effects observed
Endocrine 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
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No effects on estrus cycle.
Details on results:
HEMATOLOGY, PERIPHERAL BLOOD: All inter-group differences from controls, including those that attained statistical significance, were minor, occurred in one sex only or were without dose-relationship and were therefore considered to represent normal biological variation.
CLINICAL CHEMISTRY: All inter-group differences from controls, including those that attained statistical significance, were minor, occurred in one sex only or were without dose-relationship and were therefore considered to represent normal biological variation.
THYROID HORMONE ANALYSIS: The TSH concentrations were statistically significantly higher
than those of the controls for all groups of treated males but there was no dose response and
females were not similarly affected.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
572 mg/kg bw/day (nominal)
Based on:
other: enzyme concentrate dry matter
Sex:
male/female
Basis for effect level:
other: No adverse effects were seen at a dose with 100% of the test material.

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
It is concluded that daily oral administration of Subtilisin, batch PPA55402 to Han Wistar
rats at dose levels up to 100% of the test batch for 13 weeks was well-tolerated, with no
evidence of any adverse finding at any of the administered doses. Consequently, the
no-observed-adverse-effect level (NOAEL) was considered to be 572 mg enzyme concentrate dry matter/kg bwt/day (equivalent to 302.6 mg active enzyme protein/kg bwt/day).
Executive summary:

The purpose of this study was to assess the systemic toxic potential of Subtilisin,
batch PPA55402, an enzyme intended for use in the feed or food industry, when administered orally (by gavage) to Han Wistar rats for 13 weeks. Three groups, each comprising 10 male and 10 female rats received doses of 10, 33 or 100% of Subtilisin, batch PPA55402 (equivalent to 57.2, 188.8 or 572.0 mg enzyme concentrate dry matter/kg bwt/day, respectively). A similarly constituted control group received the vehicle (reverse osmosis water) at the same volume dose (5 mL/kg bwt/day).
During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, visual water consumption, ophthalmoscopy, hematology (peripheral blood), blood chemistry, estrous cycle, thyroid hormone, organ weight, macropathology and histopathology investigations were undertaken.

The general appearance and behaviour of the animals and sensory activity, grip strength and motor activity were unaffected by treatment. There was also no effect of treatment on body weight gain or food and water consumption. One high dose male died in Week 11 after suspected aspiration of dose and, consequently, the death of this animal was attributed to the dose administration procedure.
There were no treatment-related ophthalmoscopic findings. The haematological and blood chemistry investigation did not indicate any toxicologically significant findings. Estrous cycles at the end of the treatment period were unaffected.
Serum triiodothyronine (T3) and thyroxine (T4) concentrations were unaffected by treatment. Serum thyroid stimulating hormone (TSH) concentrations were higher than those of the controls at all dose levels in males but there was no dose response and females were not similarly affected. Consequently, the variations reported for TSH were, in the absence of any physiological response, considered to represent normal biological variation and were of no toxicological importance.
Organ weights were unaffected and there were no treatment-related macroscopic or histopathological findings.

It is concluded that daily oral administration of Subtilisin, batch PPA55402 to Han Wistar rats at dose levels up to 100% of the test batch for 13 weeks was well-tolerated, with no evidence of any adverse finding at any of the administered doses. Consequently, the no-observed-adverse-effect level (NOAEL) was considered to be 572 mg enzyme concentrate dry matter/kg bwt/day
(equivalent to 302.6 mg active enzyme protein/kg bwt/day).