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EC number: 805-156-0 | CAS number: 137010-42-5
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
![](https://echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
Repeated dose toxicity: oral
Administrative data
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 29-08-2016 to 19-10-2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Due to the similarity between the two enzymes, similar results are expected for glutamyl endopeptidase.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Version / remarks:
- Revised 3 October 2008.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
Test material
- Reference substance name:
- Active enzyme protein of trypsin (CAS no. 9002-07-7, EC no. 232-650-8, EC name trypsin, enzyme class 3.4.21.4)
- Molecular formula:
- n.a.
- IUPAC Name:
- Active enzyme protein of trypsin (CAS no. 9002-07-7, EC no. 232-650-8, EC name trypsin, enzyme class 3.4.21.4)
- 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
- 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
- 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
- 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.: PPF41399
- Expiration date of the lot/batch: 31-05-2026
- Storage condition of test material: Frosen
Constituent 1
Constituent 2
Constituent 3
Constituent 4
Constituent 5
Test animals
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on species / strain selection:
- Crl:CD(SD) rat.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Envigo RMS Limited
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 41-47 days
- Weight at study initiation: 193 to 256 g for males; 150 to 184 g for females
- Fasting period before study: None
- Housing: 5 animals of the same sex per cage
- Diet: Teklad 2014C Diet ad libitum (removed overnight before blood sampling for hematology or blood chemistry).
- Water: Ad libitum. Potable water from the public supply via polycarbonate bottles with sipper tubes.
- Acclimation period: 12 days
ENVIRONMENTAL CONDITIONS
- Temperature : 20-24°C
- Humidity : 40-70 % RH
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 22 August 2016 To: 20 Spetember 2016
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- reverse osmosis water
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test items (0, 10 and 100% active enzyme activity) were provided (deep-frozen) in a number of containers. The daily aliquots of formulation were thawed overnight in a refrigerator (2 to 8°C) if being used for dose administration on the following day, or were thawed at ambient temperature (15 to 25°C) if used for dose administration on the same day. For either case, formulations were allowed to equilibrate at room temperature before dosing and were administered within 24 hours of the commencement of thawing procedures.
VEHICLE
- Concentration in vehicle: 0, 67, 217, 435, 870, 3420 or 6841 KMTU/kg bwt/day (corresponding to 4.19, 1.11, 2.21, 4.42, 1.96 or 3.93 g TOS/kg bwt/day).
- Amount of vehicle (if gavage): constant volume of either 10 or 20 mL/kg body weight.
- Purity: Reverse osmosis water - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Dose samples were analysed according to GLP.
The purpose of this phase of the study was to determine whether the enzyme activity in the dose solutions prepared week 1, corresponded to the activity of the respective Toxbatches. - Duration of treatment / exposure:
- 4 weeks
- Frequency of treatment:
- Daily
Doses / concentrationsopen allclose all
- Dose / conc.:
- 67 other: KMTU/kg/day
- Dose / conc.:
- 217 other: KMTU/kg/day
- Dose / conc.:
- 435 other: KMTU/kg/day
- Dose / conc.:
- 870 other: KMTU/kg/day
- Dose / conc.:
- 3 420 other: KMTU/kg/day
- Dose / conc.:
- 6 841 other: KMTU/kg/day
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The highest dose (100%) is the maximum practical dose and represents administration of the enzyme, as received, at a volume dosage of 20 mL/kg body weight.
- Positive control:
- Not included
Examinations
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
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: The weight of each animal was recorded one week before treatment commenced (Day -7), on the day that treatment commenced (Day 1), weekly throughout the study (Days 8, 15, 22 and 28) and before necropsy (Days 29 or 30).
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. In Week 4 food consumption was only recorded over 6 days because of the placement of animals in metabolism cages for urine collection on the final day of the treatment period.
WATER CONSUMPTION:
- Time schedule for examinations: During Weeks -1, 1 and 3 of treatment, water consumption was recorded by weight (over a three day period on each occasion) for each cage of animals, using water bottles fitted with sipper tubes.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food. Day 29 for males and day 30 for females.
- 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)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Platelet count (Plt)
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: Blood samples were collected after overnight withdrawal of food. Day 29 for males and day 30 for females.
- Animals fasted: Yes, overnight
- How many animals: From all animals
- Parameters checked:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile Acids (Bi Ac)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Total protein (Total Prot)
Albumin (Alb)
URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected overnight at the following occasions: day 29 for males and day 30 for females.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked:
Clarity and Color (App) - by visual assessment
Volume (Vol) - using a measuring cylinder
pH - using a pH meter
Specific gravity (SG) - by direct refractometry using a SG meter
Ketones (Keto)
Bile pigments (Bili)
Urobilinogen (Urob)
Blood pigments (UBld)
Protein (T-Prot)
Glucose (T-Gluc)
Epithelial cells (Epi)
Leucocytes (WBC)
Erythrocytes (RBC)
Casts
Other abnormal components (A)
NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations: Sensory reactivity and grip strength assessments were performed (before the 1st daily dose) on all animals during Week 4 of treatment.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / : Yes - Sacrifice and pathology:
- All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. The retained tissues were checked before disposal of the carcass.
- Other examinations:
- Bone Marrow
Organ Weights
Histology - Statistics:
- The main tests used were Dunnett’s test, Barlett’s test, Williams’ test, and Fisher’s exact tests. 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:
- no effects observed
- Description (incidence and severity):
- Chin rubbing after dosing was observed at all dose levels and in both sexes, though not in the controls. This sign is commonly observed in rodent studies where the test item is administered by gavage and is associated with taste (palatability) of the test item. It was considered of no toxicological significance.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One male (No. 18) receiving 435 KMTU/kg bwt/day (Group 4) died after the first dose on Day 25. The animal appeared stressed when removed from the cage prior to dosing, showing vocalisation, having dark eyes and appearing cyanotic (blue colouration) around its nose. This animal was returned to its cage, without being dosed, and after a short interval the animal was handled and dosed with no apparent difficulty, though the coloration around the nose and dark eyes were still evident. Approximately ten minutes after dosing breathing became very irregular and a decision was made to kill the animal for welfare reasons, but the animal subsequently convulsed and died. Necropsy revealed the presence of dark fluid in the trachea and stomach and the main histopathological findings were moderate diffuse alveolar haemorrhage in the lungs, minimal periaortic haemorrhage and minimal myocardial inflammatory cell infiltrate, minimally increased extramedullary haemopoiesis and increased cellularity of the white pulp in the spleen and slight multifocal haemorrhage in the thymus. The cause of death was undetermined, but as there were no similar findings in animals receiving higher enzyme activities of SP387/TL-1, the death of this animal was not attributed to treatment.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Overall body weight gains (Days 0 to 28) were approximately 29% higher than the controls, in females receiving 6841 KMTU/kg bwt/day (Group 7). Males were unaffected. There was no effect of treatment on the body weight gain for males and females receiving 3420 KMTU/kg bwt/day or below.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Overall food consumption (Weeks 1 to 4) was approximately 13% higher than that of the controls by females receiving 6841 KMTU/kg bwt/day (Group 7) but this represented a trend that was evident during the final week of the acclimatization period and, consequently, was not attributed to treatment.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- Water consumption in Week 3 was slightly high, when compared to the controls and pretreatment values, for males receiving 6841 KMTU/kg bwt/day (Group 7). This was not a consistent finding in the three-day measurement periods and there was no dose-response, since the next highest water intake was observed at 67 KMTU/kg bwt/day (Group 2) and the high dose females were unaffected.
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- 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 biochemistry findings:
- no effects observed
- Description (incidence and severity):
- All inter-group differences from controls, including those that attained statistical significance, were minor, occurred in one sex only, were due to high/low values in a few animals within the group or were without dose-relationship and were therefore considered to represent normal biological variation.
- Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The analysis of the urine after 4 weeks of treatment revealed slightly low urinary pH at all doses and in both sexes but there was no dose-response and the majority of individual pH values were within or just below the historical control ranges at these laboratories.
- 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 organ weight analysis after 4 weeks of treatment did not identify any differences from controls that were attributable to treatment.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The macroscopic examination performed after 4 weeks of treatment revealed the presence of depressions in the glandular mucosa of the stomach of two males and four females which received 6841 KMTU/kg bwt/day (Group 7), with two of the affected females also having thickening of the non-glandular mucosa. The incidence and distribution of all other macroscopic findings were considered to be unrelated to treatment and were of the type encountered normally in Sprague-Dawley rats at these laboratories.
- Neuropathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Changes related to treatment with SP387/TL-1 were seen in the liver and stomach.
Periportal vacuolation was seen in males which received 67, 870 or 6841 KMTU/kg bwt/day (Groups 2, 5 and 7) and in females which received 67 KMTU/kg bwt/day and above (Groups 2 to 7). In females the severity was higher in the groups which received 67, 870 or 6841 KMTU/kg bwt/day (Groups 2, 5 and 7).
Epithelial hyperplasia and erosion of the non-glandular region was seen in males and females which received 6841 KMTU/kg bwt/day (Group 7).
The incidence and distribution of all other microscopic findings were considered to be unrelated to treatment. - Histopathological findings: neoplastic:
- no effects observed
Effect levels
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 3 420 other: KMTU/kg bwt/day
- Based on:
- act. ingr.
- 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 oral administration of SP387/TL-1 to Sprague-Dawley rats for 4 weeks at a range of enzyme activities up to a total daily dose of 6841 KMTU/kg bwt/day was generally well-tolerated, though at the highest dose level there were stomach erosions that were considered responses to an irritant test material and these findings were considered adverse. All other findings in this study were considered non-adverse, including the histopathological findings in the liver which occurred predominantly at 67, 870 and 6841 KMTU/kg bwt/day, corresponding to the doses that had the highest total organic solid content (approximately 4 g TOS/kg bwt/day) and the highest protein content (approximately 3 g/kg bwt/day). In view of the presence of erosions in the high dose animals the no-observed-adverse-effect level (NOAEL) was considered to be 3420 KMTU/kg bwt/day (1960 mg TOS/kg/day).
- Executive summary:
The objective of this study was to assess and compare the systemic toxic potential of a range of percentage enzyme activities of SP387/TL-1 (an enzyme used in the food industry), after oral (gavage) administration to Sprague-Dawley rats for 4 weeks. The control group received the vehicle (reverse osmosis water) at a total volume-dose of 20 mL/kg body weight (bwt). The test concentrations were 67, 217, 435, 870, 3420, and 6841 KMTU/kg bw/day.
During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food and water consumption, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken. There were no deaths associated with SP387/TL-1. The general appearance and behavior of the animals and sensory activity, grip strength and motor activity were unaffected by treatment. One male receiving 435 KMTU/kg bwt/day died on Day 25 but the death was not attributed to treatment. There was no effect of treatment upon body weight and on food and water consumption. Females receiving 6841 KMTU/kg bwt/day showed an approximately 29% higher weight gain, than the controls but this was due to their higher food intake, compared to controls, which was a trend that was present before treatment commenced. There were no treatment-related haematology or blood chemistry findings. Urine analysis revealed slightly low pH at all doses and in both sexes but there was no dose response and the findings were considered to be incidental and/or of no toxicological importance.
The statistical analysis of organ weights indicated high liver weights in males and females which received 6841 KMTU/kg bwt/day and in females which received 67 or 870 KMTU/kg bwt/day. In addition, there was a small increase of spleen weights in males which received 67 or 6841 KMTU/kg bwt/day and of heart weights in females at 6841 KMTU/kg bwt/day. There were no associated histopathological findings, the values were within concurrent or historical control ranges and these organ weight variations were considered to be due to normal biological variation and thus not related to treatment.
The macroscopic examination revealed the presence of depressions in the glandular mucosa of the stomach of two males and four females given 6841 KMTU/kg bwt/day, with two of these females also having thickening of the non-glandular mucosa. This correlated with epithelial hyperplasia and erosion of the non-glandular region of the stomach and these findings were considered related to treatment and adverse.
In the liver there was a minor but treatment-related histopathology finding to just above background levels, in the form of periportal vacuolation in males given 67, 870 or 6841 KMTU/kg bwt/day and at all doses in females; in females the extent of this finding was greatest at 67, 870 and 6841 KMTU/kg bwt/day. As these groups received the highest dose in terms of g TOS (all approximately 4 g TOS/kg bwt/day), they represented expected (and reversible) findings in response to the administration of high doses of protein and, as such, were considered toxicologically insignificant and non-adverse.
It was concluded that oral administration of SP387/TL-1 to Sprague-Dawley rats for 4 weeks at a range of enzyme activities up to a total daily dose of 6841 KMTU/kg bwt/day was generally well-tolerated, though at the highest dose level there were stomach erosions that were considered responses to an irritant test material and these findings were considered adverse. All other findings in this study were considered non adverse, including the histopathological findings in the liver which occurred predominantly at 67, 870 and 6841 KMTU/kg bwt/day, corresponding to the doses that had the highest total organic solid content (approximately 4 g TOS/kg bwt/day) and the highest protein content (approximately 3 g/kg bwt/day). All other findings in this study were considered non-adverse. In view of the presence of erosions in the high dose animals, the no-observed-adverse-effect level (NOAEL) was considered to be 3420 KMTU/kg bwt/day (1960 mg TOS/kg/day).
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