Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12-03-2010 to 12-01-2011
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:
Please state version.
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.: PPA30428
- Expiration date of the lot/batch: 30-11-2019
- Storage condition of test material: Frozen

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
Crl:CD(SD) rat.
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: 35 days
- Weight at study initiation: 118 to 145 g for males; 108 to 135 g for females
- Fasting period before study: None
- Housing: 5 animals of the same sex per cage
- Diet: The animals were allowed free access to a standard pelleted rodent diet (Rat and Mouse No. 1 Maintenance Diet), except overnight before routine blood sampling.
- Water: Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature : 19-23°C
- Humidity : 40-70% RH
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 March 2010 To: 29 June 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
water for formulation
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Prior to the commencement of treatment, and at appropriate intervals during the study, containers of test material were thawed in a refrigerator (at approximately 4°C) and divided to provide aliquots of sufficient size for the required formulation, which were then re-frozen pending use. The containers were inverted 10 times and gentle magnetic stirring was performed during the aliquoting process. The day before formulation the frozen aliquots were removed from the freezer and allowed to thaw overnight in a refrigerator (approximately 4°C). The test enzyme was prepared for administration as a series of graded concentrations in water for formulation to provide the required doses at a constant dose volume. Each formulation for the intermediate and low dose groups was prepared by gentle magnetic stirring, in order not to damage the enzyme.
VEHICLE
- Concentration in vehicle: 1, 3.3 and 10 mL/kg/day corresponding to 99, 327 and 993 mg total organic solids (TOS) /kg bw/day.
- 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:
Samples of each formulation prepared for administration in Weeks 1, 6 and 13 of treatment were analysed for achieved concentration of the test substance.
Dose samples were analysed according to GLP. The samples were transported in dry ice and then stored frozen -18°C until analysis.

Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
99 mg/kg bw/day (actual dose received)
Remarks:
mg TOS/kg bw/day
Dose / conc.:
327 mg/kg bw/day (actual dose received)
Remarks:
mg TOS/kg bw/day
Dose / conc.:
993 mg/kg bw/day (actual dose received)
Remarks:
mg 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 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: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately before dosing; Immediately after dosing on return of the animal to its cage; On completion of dosing of each group; Between one and two hours after completion of dosing of all groups; As late as possible in the working day.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded prior to the start of treatment (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 prior to the commencement of treatment (Week -1) and for each week throughout the treatment period. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.

WATER CONSUMPTION:
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. No effect was observed and, consequently, quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment started and during week 12.
- Dose groups that were examined: All animals of Groups 1 (Control) and 4 (10 mL/kg/day).

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)
Reticulocyte count (Retic)
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)
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. All animals were subject to a detailed necropsy.
HISTOPATHOLOGY: Yes: Adrenals; Brain; Femur with joint; Heart; Kidneys; Liver; Lungs; Spinal cord; Sternum; Stomach; Thyroid; Uterus.
Statistics:
The main tests used were Barlett's test, Dunnett’s test, Shirley’s test, Williams’ test, Steel's test, Fisher’s Exact 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:
effects observed, non-treatment-related
Description (incidence and severity):
Transient chin rubbing (animals seen to be pushing their jaws through the cage bedding) was seen immediately after dosing on one occasion in Week 11 in two females and one male receiving 10 mL/kg/day and in one female receiving 3.3 mL/kg/day. On the same occasion, salivation was seen at the end of dosing in one of the high dose (10 mL/kg/day) females which had exhibited post-dose chin rubbing behaviour. These are commonly-observed signs in studies where the test material is administered by gavage.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two incidental deaths occurred in this study. Female No. 76 (10 mL/kg/day) was killed on Day 5 (outside normal working hours) after showing dark eyes, abnormal (elevated) gait, deep and irregular breathing, head tremors and teeth grinding and the cause of death was identified at the histopathological examination as meningitis. Male No. 23 (10 mL/kg/day) was found dead on Day 27 and the macroscopic and histopathological examination revealed perforation of the oesophagus which was consistent with a dose-administration error.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The group mean food intake was slightly low, when compared with the Controls, during the first week of treatment for females receiving 10 mL/kg/day but the finding was largely attributable to a low value reported for cage No. 16, which housed Female No. 76. This animal was killed prematurely during the first week of treatment and it was considered likely that its poor clinical condition (the cause of death was established as meningitis) contributed to the low food consumption for this cage. Consequently, the reduction of food intake in Week 1 in these animals was not attributable to treatment.
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
Description (incidence and severity):
Red blood cell counts were marginally high and mean cell haemoglobin was marginally low, when compared with the Controls, for females receiving 10 mL/kg/day but as all individual values were within the background range for red blood cell counts and for mean cell haemoglobin. These inter-group differences were considered of no toxicological significance.
Prothrombin times were marginally longer than those of Controls for females receiving 10 mL/kg/day but as the majority (7/8) of individual values were within the concurrent control range and all results were within the background range, this finding was considered of no toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Plasma potassium concentrations were statistically significantly high (p<0.01), when compared with the Controls, in females receiving 3.3 or 10 mL/kg/day, but the differences were minor, lacked dose-relationship and the majority of values were within the background range. Consequently, this finding was considered of no toxicological significance.
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
Description (incidence and severity):
The analysis of organ weights after 13 weeks of treatment revealed, when compared with the Controls, slightly high absolute and adjusted liver weights in animals receiving 10 mL/kg/day and slightly high absolute and adjusted thymus weights in animals receiving 10 mL/kg/day and in females receiving 3.3 mL/kg/day. In both cases, statistical significance was attained only for the adjusted values. All other inter-group differences of organ weight were minor and were considered to reflect normal biological variation.
Absolute ovary weights were statistically significantly high, when compared with the Controls, for all treated groups of females but there was no dose-relationship and the group mean differences at 1 and 10 mL/kg/day were exacerbated by single incidences of enlarged ovary or unilateral periovarian sac distension reported for animal Nos. 45 (1 mL/kg/day) or 78 (10 mL/kg/day), respectively.
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:
>= 993 mg/kg bw/day (nominal)
Based on:
other: total organic solids (TOS)
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
It was concluded that oral administration of bacillolysin, batch PPA30428 to Crl:CD(SD) rats at doses up to 10 mL/kg/day for 13-weeks was well tolerated and did not result in any toxicologically significant change. Consequently, the no observed adverse effect level (NOAEL) in this study was considered to be 10 mL/kg/day (equivalent to 0.993 g TOS/kg/day).
Executive summary:

The systemic toxic potential of bacillolysin, batch PPA30428, a proteolytic enzyme, to Crl:CD(SD) rats by oral administration was assessed over a period of 13 weeks. Three groups, each comprising ten males and ten females, received bacillolysin, batch PPA30428 at doses of 1, 3.3 or 10 mL/kg/day (equivalent to 0.099, 0.327 or 0.993 g TOS/kg/day). A similarly constituted Control group received the vehicle (water for formulation) at the same volume-dose (10 mL/kg bodyweight). During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, ophthalmic examination, haematology, blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

General appearance, sensory activity, grip strength and motor activity were not affected by treatment and there were no treatment-related deaths. Chin rubbing and salivation after dosing on transient occasions in a small number of animals were considered of no toxicological significance. There were two incidental deaths at 10 mL/kg/day during the study (one female was killed in Week 1 due to meningitis and one male died in Week 4 due to an accidental dose administration error) but neither was attributable to treatment. There was no effect of treatment on bodyweight gain or food consumption. A visual assessment of water intake did not reveal any treatment-related change. There were no treatment-related ophthalmic findings. There were no toxicologically significant haematological or biochemical changes in the blood at the Week 13 investigation. Liver and thymus weights were slightly high in animals receiving 10 mL/kg/day and thymus weights were slightly high in females receiving 3.3 mL/kg/day. There were no treatment-related macroscopic or histopathological findings.

It was concluded that oral administration of bacillolysin, batch PPA30428 to Crl:CD(SD) rats at doses up to 10 mL/kg/day for 13-weeks was well tolerated and did not result in any toxicologically significant change. Consequently, the no observed adverse effect level (NOAEL) in this study was considered to be 10 mL/kg/day (equivalent to 0.993 g TOS/kg/day).