Sedolisin

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25-06-2015 to 29-10-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

3 October 2008.

Deviations:

yes

Remarks:

The only difference was that it was a 14-day instead of a 28-day study.

Principles of method if other than guideline:

The only difference was that it was a 14-day instead of a 28-day study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of sedolisin (EC no. 848318-58-1, CAS no. 814-114-0, EC name Sedolisin, Enzyme class no 3.4.21.100 )

Molecular formula:

Not available

IUPAC Name:

Active enzyme protein of sedolisin (EC no. 848318-58-1, CAS no. 814-114-0, EC name Sedolisin, Enzyme class no 3.4.21.100 )

Constituent 2

Reference substance name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available

IUPAC Name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 3

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 4

Reference substance name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Constituent 5

Reference substance name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Test material form:

liquid

Details on test material:

- Lot/batch No.: PPF38268
- Expiration date of the lot/batch: 22-april-2025

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BIONEEDS INDIA PRIVATE LIMITED
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 7 weeks
- Weight at study initiation: 111.15 g to 129.32 g for males; 104.11 g to 118.67 g for females
- Fasting period before study: None
- Housing: 3 animals of the same sex per cage
- Diet: Ad libitum Teklad Certified (2014C) Global 14% Protein Rodent Maintenance Diet - Pellet. (removed overnight before blood sampling for hematology or blood chemistry).
- Water: Ad libitum. Deep bore-well water passed through an activated charcoal filter and exposed to ultraviolet rays in Aquaguard water filter purifier was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: 7 days (29 June 2015 to 05 July 2015)

ENVIRONMENTAL CONDITIONS
- Temperature: 19.8°C to 22.9°C
- Humidity: 49-65% RH
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 July 2015 To: 19 July 2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required quantity of test material per day per group was calculated approximately, thawed by keeping al 2 to 8°C as it was frozen and made in to aliquots initially as per daily requirement. The test item aliquots were stored again as per the indication of storage condition (-20 °C) in the Certificate of Analysis. The required number of aliquot(s) were taken from the freezer and kept in refrigerator (2 to 8°C) for thawing overnight a day before formulation. The thawed aliquots were diluted with vehicle during formulation daily before dose administration. The homogeneity of vehicle/test item formulations was maintained by constant stirring on a magnetic stirrer.
VEHICLE
- Concentration in vehicle: 10% (0.5 mL/kg body weight corresponding to 65.6 mg Enzyme concentrate dry matter/kg body weight/kg body weight), 33% (1.65 mL/kg body weight corresponding to 217 mg Enzyme concentrate dry matter/kg body weight/kg body weight) and 100% (5 mL/kg body weight corresponding to 656 mg Enzyme concentrate dry matter/kg body weight)
- Amount of vehicle (if gavage): constant volume 5 mL/kg body weight.
- Purity: Distilled water

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

65.5 other: mg Enzyme concentrate dry matter/kg body weight/kg bw

Doses / concentrations 2

Dose / conc.:

217 other: mg Enzyme concentrate dry matter/kg body weight/kg bw

Doses / concentrations 3

Dose / conc.:

656 other: mg Enzyme concentrate dry matter/kg body weight/kg bw

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest dose (100%) was the maximum practical dose and represents administration of the enzyme, as received, at a volume dosage of 5 mL/kg body weight. The lower doses were selected using a ratio of approximately 3 between doses.

Positive control:

Not included

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal body weight was recorded prior to dosing on treatment day 1 and weekly thereafter (varied by -1 day) for all the animals. Fasting body weight of all the animals was recorded at their scheduled day of terminal sacrifice.

FOOD CONSUMPTION:
- Cage wise feed consumption was recorded weekly (varied by -1 day), coinciding with the body weights of the respective animals from groups. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats in each cage.

WATER CONSUMPTION:
- Time schedule for examinations: Not specified.

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmological examination was performed for all the animals before start of the treatment (during acclimatisation) and during week 2 for vehicle control and high dose group animals. As the results of examination of group G4 during week 2 did not reveal any treatment related changes, the examination was not extended to lower close groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 15
- Anaesthetic used for blood collection: Yes (isoflurane anaesthesia)
- Animals fasted: Yes, overnight (approximately 16 to 18 hours)
- How many animals: From all animals
- Parameters checked:
Haemoglobin concentration (HOB)
Haematocrit (HCT)
Erythrocyte count (RBC)
Total Leukocyte Count (WBC)
Platelet Count (PLT)
Reticulocyte count
Differential leukocyte count (DLC)
Blood Protlu-ombin time (PT) and Activated Partial Thrnmboplastin Time (APTT) were estimated by OptiClot-4 coagulation analyser.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 15
- Anaesthetic used for blood collection: Yes (isoflurane anaesthesia)
- Animals fasted: Yes, overnight (approximately 16 to 18 hours)
- How many animals: From all animals
- Parameters checked:
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
TotaI Protein
Albumin
Total bilirubin
Direct bilirubin
Glucose
Total Cholesterol
Blood Urea Nitrogen (BUN)
Creatinine
Triglycerides
Plasma Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using Prolyte Na/K/CL analyser.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. On day 15, all the animals of Group G1, G2, G3 and G4 were subjected to necropsy and detailed gross pathological examination. The animals were fasted (approximately 16 to 18 hours) overnight (water was provided ad libitum) prior to scheduled necropsy. On the day of terminal sacrifice, the body weight of all the fasted animals was recorded prior to exsanguination. The animals were euthanized using isoflurane anaesthesia followed by exsanguination. The gross pathological examination was carried out for each rat. Gross pathology examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs and tissues of each animal was conducted.

HISTOPATHOLOGY: Yes. Histopathological examination was conducted on the tissues from the vehicle control (G1) and high dose (G4) group animals. Organs and tissue samples as defined in the study plan were processed, embedded in paraffin, cut at a thickness of 4 to 6 micrometers and stained with hematoxylin and eosin. The bone marrow smear was fixed in methanol and stained with Giemsa Stain.

Statistics:

Statistical analysis was performed using SPSS software version 22, One way ANOVA. Statistical significance was noted with an * when P<0.05.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

However, statistical significant decrease in body weight gain on day 8 with respect to day 1 in G4 (males) was noted. This isolated variation is considered incidental as the mean body weights were comparable and also similar change was not observed in females.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase (P<0.05) in percent neutrophils and percent monocytes and decrease (P<0.05) in percent lymphocytes in G2 (males) and statistically significant decrease (P<0.05) in haematocrit in G3 (females) were observed. The observed variations were considered incidental and not treatment related due to lack of dose dependency or random biological variation. moreover, all of the values were within historical control range.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decrease (P<0.05) in albumin in G3 and G4 (males), statistically significant increase (P<0.05) in aspartate aminotransferase in G2, G3 and G4 (males), potassium levels in G3 and G4 (males), chloride levels in G4 (males) and statistically significant decrease (P<0.05) in glucose in G3 (females) were observed. The observed variations were considered incidental, not treatment related and due to random biological variation since the values were within the historical control range, were noted in one sex only, and/or no histopathological changes were noted in any of the treated groups.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decrease (P<0.05) in absolute thymus weight in G2 and G4 (males) and relative thymus weight in G2 (males) and statistically significant increase (P<0.05) in absolute and relative thymus and liver weights in G3 (females) were observed. All the observed variations were considered incidental and not treatment related because of lack of dose dependency and/or there were no associated macroscopic and microscopic abnormalities noted.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 656 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 so NOAEL was the highest dose administered.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

It was concluded that the No Observed Adverse Effect Level (NOAEL) of the test item Sedolisin, batch PPF38268 was found to be the high dose group of 5 mL/kg body weight/day of 100% v/v, corresponding to 656 mg enzyme concentrate dry matter/kg body weight/day when administered for a period of 14 consecutive days by oral (gavage) route to Sprague Dawley rats under the experimental conditions and the doses employed.

Executive summary:

Sedolisin, batch PPF38268 was evaluated to determine the toxic potential when administered to Sprague Dawley rats through oral route for 14 consecutive days. This study provides information on any major toxic effects, target organs and an estimate of the No Observed Adverse Effect Level (NOAEL).

A total or 48 (24 males + 24 females) Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 6 males and 6 females. The animals in G1 group were administered with vehicle (distilled water), the animals in G2, G3 and G4 groups were administered with test item Sedolisin, batch PPF38268 at the dose levels of 10, 33 and 100%, v/v, respectively. The vehicle and test item formulations were administered at the dose volume of 5 mL/kg body weight corresponding to 656 mg enzyme concentrate dry matter/kg bpdy weight/day.

The dose formulations were stable in distilled water for 24 hours at room temperature as per the sponsor specification. The frozen test item was thawed at 2 to 8°C overnight before formulation preparation. The formulations were prepared freshly before dose administration.

All animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight, percent change in body weight and feed consumption. Ophthalmological examination was carried out during week 2 for G1 and G4 group animals. Hematology, clinical chemistry analysis, gross pathology and organ weighing were performed on day 15 for all the animals. Histopathological examination was conducted on the organs/tissues from the vehicle control (G1) and high dose (G4) group animals.

The animals did not reveal any clinical signs of toxicity and mortality or morbidity throughout the study period. No treatment related changes in body weight, body weight gain with respect to day 1, feed consumption, ophthalmoscopic examination, hematology, clinical chemistry, organ weights (both absolute and relative) were observed. There were no gross pathological changes observed at any of the doses tested in either sex. No treatment related histopathological findings were noticed in the study.

Based on the results of the experiment it was concluded that the No Observed Adverse Effect Level (NOAEL) of the test item Sedolisin batch PPF38268 was found to be high dose group of 5 mL/kg body weight/day of 100% v/v, corresponding to 656 mg Enzyme concentrate dry matter/kg body weight/day when administered for a period of 14 consecutive days by oral (gavage) route to Sprague Dawley rats under the experimental conditions and the doses employed.