Mucorpepsin

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21-10-1991 to 21-03-1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of mucorpepsin (CAS no.148465-73-0, EC no 642-981-3, enzyme class 3.4.23.23)

Molecular formula:

Not applicable

IUPAC Name:

Active enzyme protein of mucorpepsin (CAS no.148465-73-0, EC no 642-981-3, enzyme class 3.4.23.23)

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:

solid: particulate/powder

Details on test material:

- Lot/batch No.: PPR 3583
- Expiry data of the lot/batch: 2002-12-15

Test animals

Species:

rat

Strain:

other: CD strain

Details on species / strain selection:

Obtained from Charles River (UK) Limited, Margate, Kent, England.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 21 to 28 days of age
- Weight at study initiation: Between 99 and 145 g eight days after arrival.
- Fasting period before study: Not specified.
- Housing: The rats were housed five of one sex per cage. The cages used were Type TRIS, which were made of a stainless steel body measuring 51 x 38 x 20 cm with a stainless steel mesh lid and floor. The cages were suspended above absorbent crepe paper which was changed three times a week.
- Diet: A powdered rodent diet, Laboratory Animal Diet No. 2 was available ad libitum except when urine was being collected and overnight before routine blood sampling.
- Water: Water taken from the public supply (Suffolk Water Company, Lowestoft, England), was available ad libitum via polycarbonate bottles fitted with sipper tubes, except when urine was being collected.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21⁰C
- Humidity (%): 55%
- Air changes (per hr): At least 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12-hour light : 12-hour dark cycle.

IN-LIFE DATES: From: 18 December 1991 To: 25 March 1992

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The test material was administered continuously via the diet throughout the treatment period. The dietary concentration was maintained at a constant level for each group throughout the treatment period. Animals did not have access to mixed diet beyond the end of its shelf-life as determined by the stability test. Control animals received untreated diet at the same frequency, and from the same batch, as treated animals.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The rennilase was incorporated into the ground diet to provide the required dietary concentrations. Initially the test substance was mixed to achieve a concentration of 10000 ppm. This diet was divided into two aliquots. One was used for treatment of the high dosage group and the other for preparation of the diet for the remaining treatment groups by a serial dilution process. Batches of the test diets were prepared each week and issued in sealed polyethyl ene bags. The unused residue at the end of each week was discarded.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The identity, strength, purity and composition, or other characteristics of the batch were analysed by Novo Nordisk (= Novozymes A/S since 2000).

Duration of treatment / exposure:

All animals were treated for at least 13 weeks.
Treatment, and the recording of serial observations, continued for all surviving animals throughout the necropsy period.

Frequency of treatment:

The test material was administered continuously via the diet throughout the treatment period. The dietary concentration was maintained at a constant level for each group throughout the treatment period. Animals did not have access to mixed diet beyond the end of its shelf-life as determined by the stability test. Control animals received untreated diet at the same frequency, and from the same batch, as treated animals.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 000 ppm

Remarks:

610.8 and 695 mg enzyme concentrate dry matter/kg/day for males and females, respectively

Doses / concentrations 2

Dose / conc.:

2 000 ppm

Remarks:

124 and 138.3 mg enzyme concentrate dry matter/kg/day for males and females, respectively

Doses / concentrations 3

Dose / conc.:

400 ppm

Remarks:

25 and 29.3 mg enzyme concentrate dry matter/kg/day for males and females, respectively

No. of animals per sex per dose:

20

Control animals:

yes

Details on study design:

- Dose selection rationale: The dietary route was selected to accord with the major potential route of exposure in manufacture and use.
The doses were selected based on results from a dose range-finding study, where the highest dose were considered suitable for this 13-week study and concentrations of 400 and 2000 ppm were selected using a geometric ratio of 5.

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: Rats were inspected at least twice daily for evidence of reaction to treatment or ill-health. In addition a more detailed weekly examination, which included palpation, was performed on each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed during the acclimatisation period, on the day that treatment commenced, at weekly intervals throughout the treatment period and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. The weight of food supplied to each cage, that remaining and an estimate of the amount spilled was recorded for each week throughout the treatment period. From these records the mean weekly consumption per animal was calculated for each cage. Weekly food conversion efficiencies were calculated for each week of the treatment period.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. The achieved dosages averaged 25, 124 and 610.8 mg enzyme concentrate dry matter/kg/day for males and 29.3,138.3 and 695 mg enzyme concentrate dry matter/kg/day for females treated at 400, 2000 and 10000 ppm respectively.

FOOD EFFICIENCY: The weekly group mean values presented were calculated by first deriving the weekly cage values. These were calculated from the bodyweight gains and the total weight of food consumed in the cage. Weekly group means were derived from unrounded cage values. Overall group mean values were calculated from the weekly group mean values presented.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Animals and their cages were observed daily for any changes that might suggest a treatment-related effect on body fluids balance. No quantitative measurements were made.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before commencement of treatment both eyes of all rats were examined by means of an indirect ophthalmoscope, after the instillation of 0.5% tropicamide. After 12 weeks of treatment all animals from Groups 1 and 4 were similarly examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 12 weeks of treatment blood samples were obtained from the ten male and ten female animals with the highest animal numbers in each group, after overnight starvation.
- Anaesthetic used for blood collection: Yes. Halothane/nitrous oxide anaesthesia.
- Animals fasted: Yes
- How many animals: 80
- Parameters checked included: Packed cell volume (PCV), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Total and differential leucocyte count (WBC) Platelet count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the same time and using the same animals as for peripheral haematology further blood samples were taken and collected into lithium heparin as anticoagulant.
- Animals fasted: Yes
- How many animals: 80
- Parameters checked included: Alkaline phosphatase activity, Alanine amino-transferase activity, Aspartate amino-transferase activity, Ornithine carbamyl transferase activity, Ganuna-glutamyl transpeptidase activity, Urea concentration, Creatine phosphokinase activity, Glucose concentration, Total bilirubin concentration, Total protein concentration, Electrophoretic protein fractions, Sodium (Na), potassium (K), chloride (Cl), calcium (Ca) and inorganic phosphorus (P) concentrations.

URINALYSIS: Yes
- Time schedule for collection of urine: After 11 weeks of treatment overnight urine samples were collected from the ten male and ten female animals with the highest animal numbers in each group.
- Metabolism cages used for collection of urine: Yes. Animals were deprived of water for approximately 12.30 hours. At approximately 17.00 hours each animal was placed in an individual metabolism cage without food and water, and urine was collected until approximately 09.00 hours the following day.
- Animals fasted: Yes.
- Parameters checked in table were examined. The individual samples were examined for: Appearance, volume, pH, Specific gravity, Protein, Total reducing substances, Glucose, Ketones, Bilirubin, Urobilinogen, Nitrite, Blood.

Sacrifice and pathology:

Organ weights:
The weight following organs were recorded: Adrenals, brain, epididymides, heart, kidneys, liver, lungs with mainstem bronchi, ovaries, pituitary, prostate, salivary glands, seminal vesicles, spleen, testes, thymus, thyroid with parathyroid and uterus with cervix.

Histology:
Tissue samples from the animals were dehydrated, embedded in paraffin wax, sectioned at approximately five micron thickness and stained with haematoxylin and eosin. The thyroid section included parathyroid tissue whenever possible. Tissue from auricular and ventricular regions of the heart were prepared. Sections from two lobes of each of the liver and lungs were similarly processed. The brain was sectioned to include the cerebellum, cerebral cortex and medulla. The spinal cord was prepared in transverse section at the cervical and thoraco-lumbar levels.

Statistics:

Standard deviations were calculated, as considered appropriate, using the sample statistic. The significance of inter-group differences in haematology, blood chemistry and urinalysis (volume, specific gravity and pH only) was assessed by Student's 't'-test using a pooled error variance. Statistical significances for reticulocyte, eosinophil, basophil, monocyte and large unstained cell counts are not reported as these data are not normally distributed. For organ weights and bodyweight changes, homogeneity of variance was tested using Bartlett's test. Whenever this was found to be statistically significant a Behrens-Fisher test was used to perform pairwise comparisons, otherwise a Dunnett's test was used. Inter-group differences in ophthalmoscopy, macroscopic pathology and histopathology were assessed using the Fisher Exact test which was applied as a two-tailed test. Unless stated, group mean values were not significantly different from those for the controls (p>0.05).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Bodyweight gains were unaffected by treatment. The overall bodyweight gains of males receiving 400 ppm and females receiving 2000 ppm were slightly higher than those of the controls. This was considered to reflect the slightly high food consumption seen in these groups and, in the absence of any dosage-relationship, was not attributed to treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no inter-group differences in food consumption which could be attributed to treatment. Slightly high food consumption when compared to the controls was observed in males receiving 400 ppm and in females receiving 2000 ppm. However, this was considered to be a chance inter-group difference due to normal biological variation and was not attributed to treatment.

Food efficiency:

no effects observed

Description (incidence and severity):

The food conversion efficiency was unaffected by treatment.

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

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related ocular lesions.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no haematological changes that were attributable to treatment with rennilase. A number of inter-group differences attained statistical significance when compared with controls (p<0.05) but these were minor in nature or lacked dosage-relationship and were considered to represent normal biological variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in the plasma after 12 weeks that could unequivocally be ascribed to treatment with rennilase. After 12 weeks of treatment the plasma glucose concentrations of treated animals tended to be marginally higher than those of the controls. This change was considered to be due to slightly lower than expected values for the controls and, consequently, plasma glucose concentration were considered to have been unaffected by treatment. Several other inter-group differences attained statistical significance when compared with the controls (p<0.05) but these were minor in nature or lacked dosage relationship.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The composition of the urine was unaffected by treatment with rennilase. After 11 weeks of treatment marginally higher urinary volumes than in the controls were seen in males receiving 10000 ppm. This inter-group difference was small and, in the absence of any similar effect in females, was not attributed to treatment.

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):

There were no inter-group variations in organ weight after 13 weeks which were attributed to treatment. The bodyweight-relative liver weights of females treated at 10000 ppm was significantly higher than those of the controls (p<0.05). The inter-group difference was, however, very small and was not considered to be toxicologically significant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic changes after 13 weeks of treatment that were attributed to the administration of rennilase.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

After dietary administration of rennilase, an enzyme, to CD rats for 13 weeks, chronic myocarditis was observed at a higher incidence than in the controls in males that received 10000 ppm, but otherwise there was no evidence of systemic toxicity. This change is a normal spontaneous finding in ageing rats but it is also known to be exacerbated by a number of materials. The reason for the absence of a similar effect in females is unknown. As such, this finding was considered to be of little toxicological significance.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOEL

Effect level:

>= 124 mg/kg bw/day (actual dose received)

Based on:

other: enzyme concentrate dry matter

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Chronic myocarditis was observed at a higher incidence than in the controls in males that received 10000 ppm. This change is a normal spontaneous finding in ageing rats but it is also known to be exacerbated by a number of materials.

2

Key result

Dose descriptor:

NOEL

Effect level:

>= 138.3 mg/kg bw/day (actual dose received)

Based on:

other: enzyme concentrate dry matter

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: The bodyweight-relative liver weights of females treated at 10000 ppm was significantly higher than those of the controls.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Dietary administration of rennilase to CD rats for 13 weeks produced effects in the heart of the high dose males, but otherwise there was no evidence of systemic toxicity.

Chronic myocarditis was observed at a higher incidence than in the controls in males that received 10000 ppm (610.8 mg enzyme concentrate dry matter/kg/day). This change is a normal spontaneous finding in ageing rats but it is also known to be exacerbated by a number of materials. The reason for the absence of a similar effect in females is unknown. As such, this finding is considered to be of little toxicological significance.

In conclusion, the administration of rennilase to CD rats at a level of 10000 ppm (610.8 mg enzyme concentrated dry matter/kg/day for males and 695 mg enzyme concentrate dry matter/kg/day for females) was associated with only minor evidence of toxicity. The no-effect level on this study was 2000 ppm (124 mg/kg/day for males and 138.3 mg/kg/day for females).

Executive summary:

Three groups of 20 male and 20 female CD rats received Rennilase, batch PPR 3583 in the diet at concentrations of 400, 2000 or 10000 ppm for 13 weeks. A similarly constituted control group received untreated diet.

There were no deaths and no signs related to treatment. Bodyweight gains, food consumption and the efficiency of food utilisation were unaffected by treatment. The achieved dosages averaged 25, 124 and 610.8 mg enzyme concentrate dry matter/kg/day for males and 29.3,138.3 and 695 mg enzyme concentrate dry matter/kg/day for females treated at 400, 2000 and 10000 ppm, respectively. There were no treatment-related ocular lesions. The composition of the blood and urine was unaffected by treatment. There were no organ weight changes or macroscopic abnormalities which were considered to be related to treatment.

Chronic myocarditis was observed at a higher incidence than in the controls in males that received 10000 ppm (610.8 mg enzyme concentrate dry matter/kg/day). This change is a normal spontaneous finding in ageing rats but it is also known to be exacerbated by a number of materials. The reason for the absence of a similar effect in females is unknown. As such, this finding is considered to be of little toxicological significance.

In conclusion, the administration of rennilase to CD rats at a level of 10000 ppm (610.8 mg enzyme concentrate dry matter/kg/day for males and 695 mg enzyme concentrate dry matter/kg/day for females) was associated with only minor evidence of toxicity. The no-effect level on this study was 2000 ppm (124 mg enzyme concentrate dry matter/kg/day for males and 138.3 mg enzyme concentrate dry matter/kg/day for females).