Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Active enzyme protein of Aspergillopepsin I (EC no. 232-796-2, CAS no. 9025-49-4, EC name: Proteinase, Aspergillus acid, Enzyme Class no. 3.4.23.18)
Molecular formula:
Not applicable, see remarks
IUPAC Name:
Active enzyme protein of Aspergillopepsin I (EC no. 232-796-2, CAS no. 9025-49-4, EC name: Proteinase, Aspergillus acid, Enzyme Class no. 3.4.23.18)
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.: AFP501K1

Test animals

Species:
rat
Strain:
other: Ntac:SD
Details on species / strain selection:
The rat was selected as the test model because of its proven suitability in this type of study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic M&B A/s, Ejby, Denmark
- Females (if applicable) nulliparous and non-pregnant: not reported
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: mean of 195.1-200.4 g for males and 160.2-166.5 g for females
- Fasting period before study: no
- Housing: Transparent polycarbonate cages (floor area: 1500 cm2 – height 21 cm) with 2 in each cage, males and females separated.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: Analyses of diet for major nutritive components and relevant possible contaminants were performed regularly. Analyses for relevant possible contaminants were performed regularly on the drinking water prior to acidification.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±3°C
- Humidity (%): 55±15%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: 1 mL stock solution contained 124.7 mg total protein. The test item (as stock solution) was stored frozen at approximately -18°C until use. Before each use, each bottle of the stock solution was thawed to divide the contents into portions suitable for weekly preparation. The stock solution was thawed overnight in a refrigerator. Before dividing the contents of the original stock bottle into portions and before preparation of the dosing formulations, the stock solution was stirred gently for at least 10 minutes on a magnetic stirrer.

Group 1 was dosed with vehicle (50 mL water for injection). The dose formulations for the test groups (Groups 2, 3, and 4) were prepared weekly by diluting the test item (stock solution) with vehicle. Group 2 was 0.05 mL stock solution plus 4.95 mL water for injection. Group 3 was 0.10 mL stock solution plus 4.90 mL water for injection. Group 4 was 0.25 mL stock solution plus 4.75 mL water for injection. According to the Sponsor, the prepared dosing formulations were stable for more than 7 days when stored at refrigerator temperature in the dark and for more than 6 hours at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In Weeks 1, 6, and 13, duplicate (2) samples of the four dose formulations were taken into a 1.8 mL Cryotube and stored frozen at approximately -18°C and subsequently sent with dry ice for analysis. The samples were analysed.

The total protein results were similar within a satisfactory range to the expected concentrations.
Duration of treatment / exposure:
13-Weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
6.25 other: mg total protein/kg
Dose / conc.:
12.5 other: mg total protein/kg
Dose / conc.:
31.25 other: mg total protein/kg
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected by the Sponsor

Dose formulations for the test groups (Groups 2-4) were kept on a magnetic stirrer during treatment.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily during the morning hours. An additional morbidity/mortality check was performed in the afternoon.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Beginning prior to start of treatment, detailed clinical observations were performed outside the home cage once per week at similar times. Signs to be recorded included, but were not limited to changes in skin/fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, sterotypies or bizarre behaviour were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed one week before start of dosing (Day -7), on the first day of treatment (Day 1) and weekly thereafter. Also the weight at necropsy was recorded.

FOOD CONSUMPTION: Yes
- Time schedule: Starting Day 1, the consumption of food was recorded weekly for each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: all animals examined before start of treatment. All animals in groups 1 and 4 examined before termination of treatment.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Before termination of treatment
- Anaesthetic used for blood collection: Yes (CO2/O2 anaesthesia)
- Animals fasted: Not specified
- How many animals: all
- Parameters examined: Haemoglobin (Hb), red blood cell count (RBC), haematocrit (HT), mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), white blood cell count (WBC), differential leucocyte count (NEUTRO, LYMPHO, EOS, BASO, MONO), platelet count (PLT), activated partial thromboplastin time (APTT), prothrombin time (Pt), fibrinogen (Fib)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Before termination of treatment
- Animals fasted: Not specified
- How many animals: all
- Parameters examined: Alanine aminotransferase (ALAT), aspartate amino transferase (ASAT), alkaline phosphatase (ALKPH), bilirubin (total) (BILI), gamma-glutamyl transferase (GGT), cholesterol (CHOL), triglycerides (TRIG), carbamide (UREA), creatinine (CREAT), glucose (GLUC), sodium (Na), potassium (K), calcium (Ca), Magnesium (Mg), inorganic phosphorus (P), chloride (Cl), protein (total) (PROTEIN), albumin (ALB), globulin, albumin/globulin (ALB/G) ratio

URINALYSIS: Yes
- Time schedule for collection of urine: Before termination of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: volume, sodium (Na), potassium (K), chloride (Cl), specific gravity (SG), pH, colour (COLOUR), protein (PROTEIN), leucocytes (LEUC), blood (BLOOD), glucose (GLUCOSE), ketones (KETONES), bilirubin (BILI), urobilinogen (UROBIL). Microscopic examination of spun sediment was performed. A 40x magnification was used. For the various findings, the incidence was described in the following ways: - “no trace” = no trace in 2-3 visual fields; (+) “trace” = a few in 2-3 visual fields; + “slight” = a few in each visual field; ++ “moderate” = several in each visual field; +++ “marked” = numerous in each visual field. The elements examined were erythrocytes, leukocytes, epithelial cells, crystals, urates, hyaline and granular caste and bacteria

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: One occasion during the last 2 weeks of the study
- Dose groups that were examined: All
- Battery of functions tested: Reactivity to different types of stimuli, grip strength, and motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table); Paired organs were weighed together. The relative organ weights were calculated for each animal. All tissues were initially fixed in phosphate buffered neutral 4% formaldehyde with the exception of the eyes (Davidsons’s fixative) and tested (Bouin’s fixative). The fixative for the long term preservation was phosphate buffered neutral formaldehyde for all tissues. The lungs were infused with fixative at necropsy

HISTOPATHOLOGY: Yes (see table); Specimens were embedded in paraffin and cut at a nominal thickness of approximately 5 µm, stained with haematoxylin and eosin and examined under a light microscope. Paired organs were processed together. Histological alterations were graded on a 5 grade system: Grade 1 – minimal/very few/very small; Grade 2 – slight/few/small; Grade 3 – moderate/moderate number/moderate size; Grade 4 – marked/many/large; Grade 5 – massive/extensive number/extensive size; Present – finding present/severity not scored. All organs from all control and high dose animals were examined microscopically. Submandibular lymph nodes with macroscopic visible signs of accumulation of blood due to blood sampling from the ipsilateral orbital venous plexus were fixed but not processed histologically. Both eyes were fixed but only the eye opposite the side used for blood sampling was examined microscopically.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Thereafter each continuous variable was tested for homogeneity of variance with Bartlett’s test. If the variance was homogenous, analysis of variance was carried out for the variable. If any significant differences were detected, possible inter-group differences were assessed with Dunnett’s test (comparing treated groups with a control group). If the variance was heterogeneous, each variable was tested for normality by the Shapiro-Wilk method. In case of normal distribution, possible inter-group differences were identified with Student’s t-test. Otherwise the possible inter-group differences were assessed by Kruskal-Wallis’s test. If any significant inter-group differences were detected, the subsequent identification of the groups was carried out with Wilcoxon Rank-Sum test. Ranked type of urine analysis data was analysed with Wilcoxon Rank-Sum test. For all tests, the level of significance was defined at p<0.05.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Sporadic findings (primarily hair loss, discharge from the eyes and small wounds) reported in the animals throughout the study were considered to be within what could be expected in a study of this duration and therefore not associated with treatment.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
All animals gained expected body weight during the study and no treatment-related effects were observed on body weights or body weight gain. On day 43 the female animals at 6.25 mg total protein/kg had a statistically significant decreased body weight when compared to the females of the control group. Since this finding was sporadic, without clear dose dependency and without a similar tendency in the opposite sex, it was considered to be incidental.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In week 3, the females at 6.25 mg total protein/kg had statistically significantly lower food consumption when compared to the females of the control group. Since this finding was sporadic, without clear dose dependency and without a similar tendency in the opposite sex, it was considered to be incidental.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At termination, a statistically significant decrease for sodium was seen in males at 12.5 mg total protein/kg compared to the control group. As this was seen with the opposite tendency in the females, and seen in the intermediate dose level only, it is not considered to be attributable to treatment.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
At termination, a statistically significant increase for chloride was seen in males at 12.5 total protein/kg compared to the control group. As this was seen with the opposite tendency in the females, and seen in the intermediate dose level only, it is not considered to be attributable to treatment.
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At termination, in the males, the absolute and relative liver weight was statistically significant decreased and the relative testes weight was statistically significant increased compared to the control group. However, since these weight changes were within the 95% confidence interval of the test facility historical control data (liver weights males (absolute): 13052-20132, relative: 2.905-3.854. Testes weight (relative): 0.663-0.981) and because no liver or testes-related findings were observed at the microscopic examination in this study, the changes were considered to be of no toxicological importance.
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:
NOEL
Effect level:
31.25 other: mg total protein/kg (35.81 mg TOS/kg)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
13-Week NOEL: 31.25 mg/total protein/kg (35.81 mg TOS/kg) (highest dose tested)
Executive summary:

The test substance was administered daily by oral gavage to rats for 13 weeks. The study was conducted in accordance with OECD Guideline 408. The rat was selected as the test model because of its suitability in this type of study. Oral treatment was chosen to comply with the intended route of exposure in humans. The doses were selected by the Sponsor. Eighty rats (40 males and 40 females) were used in the study. The animals were allocated to four groups (10/sex/group) and were treated once daily by oral gavage for 91 days with water for injection (control), 6.25, 12.5, or 31.25 mg total protein/kg bw/day. One mL stock solution contained 124.7 mg total protein (142.9 mg TOS). The dose volume was 5 mL/kg bw. Clinical signs were recorded daily. Detailed clinical observations were performed once weekly. During Week 12 and 13 of the study, the animals were examined for sensory reactivity, grip strength, and motor activity. Ophthalmoscopy was performed on all animals before start of treatment, and on the animals at 6.25 and 31.25 mg total protein/kg during Week 13 of the study. Body weight and food consumption were recorded weekly. Before termination of treatment, blood samples were taken for haematology and clinical chemistry, and urine was collected for urinalysis. The animals were kidded and subjected to a macroscopic necropsy. Specified organs/tissues were weighed, fixed and prepared for a histopathological examination.

No treatment-related findings were recorded at the clinical and behavioural examinations, on food consumption, body weights or at the ophthalmoscopic examination. No treatment-related findings were observed on the parameters for serum biochemistry, urinalysis, and on organ weight. Necropsy and the following microscopic examination revealed no treatment-related effects. Daily oral administration of the test substance to rats at doses of 6.25, 12.5, and 31.25 mg total protein/kg (corresponding to 7.16, 14.32, and 35.81 mg TOS/kg, respectively) for 13 weeks was well tolerated and did not produce any toxicologically significant changes. Consequently, the no-observed-effect level (NOEL) in this study was considered to be 31.25 mg total protein/kg (35.81 mg TOS/kg).