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EC number: 232-983-9 | CAS number: 9075-68-7
- 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
Endpoint summary
Administrative data
Description of key information
The repeated dose oral toxicity of pullulanase has been tested. The repeated dose inhalation and dermal toxicity were waived based on exposure considerations and the properties of the substance. The repeated dose oral toxicity was a subchronic toxicity test conducted according to OECD guideline 408 , and in compliance with GLP. The conclusion was that the No Observed Adverse Effect Level (NOAEL) in rats was the highest dose level tested - 851 mg/kg bw/day based on TOS (total organic solids).
Key value for chemical safety assessment
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 12 December 2012 - 11 September 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- The purpose of the study was to satisfy regulatory demands because the enzyme is also used for production of food in EU.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Version / remarks:
- revised 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River, UK, Ltd
- Age at study initiation: 42 to 48 days
- Weight at study initiation: 219-276 g for males; 166-222 g for females
- Fasting period before study: None
- Housing: 5 of same sex per cage
- Diet : Standard pelleted rodent diet ad libitum
- Water : Tap water ad libitum
- Acclimation period: 13 days
ENVIRONMENTAL CONDITIONS
- Temperature : 19-23°C
- Humidity : 40-70 % RH
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 2013-01-29 To: 2013-01-05 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 10, 33 and 100%, corresponding to 85, 281 and 851 mg total organic solids (TOS) per kg body weight per 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:
- Dose samples were analysed according to GLP.
Analysis of achieved concentration with regard to the enzyme activity was performed on samples taken once during weeks 1, 6 and 13. No significant differences were found between weeks and no significant differences were found between the achieved concentration for the high dose group and the 100% undiluted tox-batch, demonstrating satisfactory formulation. - Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- Daily
- Dose / conc.:
- 85 mg/kg bw/day (nominal)
- Remarks:
- 1.0 mL/kg/day, equivalent to 0.085 g total organic solids (TOS)/kg/day nominal in water
- Dose / conc.:
- 281 mg/kg bw/day (nominal)
- Remarks:
- 3.3 mL/kg/day, equivalent to 0.281 g total organic solids (TOS)/kg/day nominal in water
- Dose / conc.:
- 851 mg/kg bw/day (nominal)
- Remarks:
- 10.0 mL/kg/day, equivalent to 0.851 g total organic solids (TOS)/kg/day nominal in water
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The doses used in this study were selected on the basis of results from studies performed on other similar enzyme preparations. The highest dose (10 mL/kg/day) was the maximum practical dose and represents administration of the enzyme, as received, at a volume-dose of 10 mL/kg body weight, which is the maximum practical volume-dose for repeat dose oral administration. The lower doses were selected using an approximate ratio of 3.3 between doses.
- Positive control:
- Not included
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Pre-dose and twice daily during the first week of treatment, twice daily during Weeks 2 to 5 and weekly thereafter, detailed observations were recorded
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
FOOD CONSUMPTION:
- Food consumption for each cage, i.e. sum of five animals: Yes weekly throughout the study
WATER CONSUMPTION: Yes
- Time schedule for examinations: Recorded by weight (over a 3 day period on each occasion) weekly throughout the study for each cage
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-treatment and during week 12
- Dose groups that were examined: Control and highest dose group
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Isoflurane
- 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)
Total white cell count (WBC)
Differential WBC count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)
Prothrombim time (PT)
Activated partial thromboplastin time (APTT)
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)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Total protein (Total Prot)
Albumin (Alb)
Albumin/globulin ratio (A/G Ratio)
URINALYSIS: Yes
- Time schedule for collection of urine: Week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked:
Clarity and colour (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)
Bilirubin/bile pigments (Bili)
Blood pigments (UBld)
Protein (Prot)
Creatinine (U-Creat)
Glucose (U-Gluc)
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, full macroscopic examination
HISTOPATHOLOGY: Yes (Full list: Animals from Control and high dose group, Abnormalities only from mediate and low dose groups). - Other examinations:
- FAECAL ANALYSIS: No
Weight of individual organs: Yes
- Time schedule for collection of organs: At necropsy - Statistics:
- Statistical evaluation of grip strength, motor activity, bodyweight, haematology, blood chemistry, urinalysis, organ weights and any data derived from these was performed. The level of probability chosen as significant was p<0.05.
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- no effects observed
- 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):
- All differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation.
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- All differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation
- Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- It was considered that the high urinary volumes observed and low urinary pH may be due to a physiological adaptive response to the high salt content (> 4%).
- Behaviour (functional findings):
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- not specified
- Details on results:
- HAEMATOLOGY:
All differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included increased reticulocyte and lymphocyte counts and small prolongations of activated partial thromboplastin times at all doses in females. For reticulocyte counts the differences from controls were small, none of the individual values was outside the background range (0.097x10^12 to 0.253x10^12/L; n=18) and there was no effect upon other erythrocyte parameters. The increased lymphocyte count at all doses in females was not dose-related and none of the individual values was abnormal. In addition, the group mean value for the controls (3.35x10^9/L) was well below the mean of the background data (6.85x10^9/L; n=307) and this finding was therefore a consequence of lower than expected values in the control group. The small prolongation of activated partial thromboplastin time similarly lacked dose relationship and all values were well within the background range (11.1 to 21.2 seconds; n=308). Consequently, each of these trends was considered fortuitous.
CLINICAL CHEMISTRY
All differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the low creatinine concentrations in males receiving 100% Pullulanase, PPY34283, since the group mean value of the control group was raised by one unusually high individual value (No. 6: 76 μmol/L) and all individual values in the high dose group were within the background range (90 percentile range 27 to 46 μmol/L; n=299). They also included the low potassium concentrations in males receiving 100% Pullulanase, PPY34283, where all values were within the background range (90-percentile range 4.0 to 6.1 mmol/L; n=289).
URINALYSIS:
The urinalysis investigation in Week 13 revealed, when compared with the controls, high urinary volumes associated with slightly low specific gravity at all doses in males, but particularly those receiving 33 or 100% Pullulanase, PPY34283 where statistical significance was attained. In males and females given 100% Pullulanase, PPY34283 there was a clear reduction of urinary pH. It was considered that this may be due to a physiological adaptive response to the high salt content of the Toxbatch (> 4%). - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 851 mg/kg bw/day (nominal)
- Based on:
- other: total organic solids (TOS)
- Sex:
- male/female
- Basis for effect level:
- other: see 'Remark'
- Critical effects observed:
- not specified
- Conclusions:
- The No Observed Adverse Effect Level (NOAEL) in rats was 10 mL of the undiluted pullulanase/kg body weight/day equivalent to 851 mg Total Organic Solids (TOS)/kg body weight/day.
- Executive summary:
The repeated dose oral toxicity study was a subchronic toxicity test conducted according to OECD guideline 408 (revised 1998), and in compliance with GLP. The purpose of the study was to satisfy regulatory demands because the enzyme is used for production of food in EU.
In conclusion, oral administration (by gavage) of pullulanase to rats at dosages of up to the highest dose level tested, equivalent to 10 mL of the undiluted test material/kg body weight/day or 851 mg Total Organic Solids (TOS)/kg body weight/day for thirteen weeks was well-tolerated and did not produce any toxicologically significant changes.
Consequently, the No Observed Adverse Effect Level (NOAEL) was considered to be the highest dose level administered, equivalent to 10 mL of the undiluted pullulanase/kg body weight/day or 851 mg Total Organic Solids (TOS)/kg body weight/day.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 851 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- Toxicological data has been generated within the enzyme producing industry during the last 40 years. Substantial documentation on the safety of the production strains has been generated, and the enzyme test materials are thoroughly characterized. High quality studies for all relevant endpoints for in vivo studies as well as in vitro studies show that industrial enzymes from well-known and well characterized production strains have very similar safety profiles across the catalytic activities. Read-across can therefore be applied for the majority of toxicological endpoints. The database can therefore be considered of high quality.
Repeated dose toxicity: inhalation - systemic effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: inhalation
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- other:
- Critical effects observed:
- not specified
Reference
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: inhalation - local effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: inhalation
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- other:
- Critical effects observed:
- not specified
Reference
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - systemic effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
The repeated dose oral toxicity of pullulanase has been tested, while the repeated dose inhalation and dermal toxicity were waived.
- The dermal study was waived because of the low likelihood of absorption of enzymes through the skin due to the physico-chemical properties of the enzyme protein.
- The inhalation study was waived because exposure is too low to exert any toxicity. Potential exposure by inhalation to an amount of enzyme, which is toxicologically relevant, is unrealistic due to the stringent work practices and the formulation of enzymes, enforced because of the risk of sensitization by inhalation.
- The repeated dose oral toxicity was a sub-chronic toxicity test conducted according to OECD guideline 408 (adopted 1998), and in compliance with GLP. The conclusion was that the No Observed Adverse Effect Level (NOAEL) in rats was the highest dose level tested, equivalent to 10 mL of undiluted test material/kg bw/day or 851 mg Total Organic Solids (TOS)/kg bw/day.
Based on repeated dose oral and weight of evidence, pullulanase does not exert any repeated dose oral, dermal or inhalation toxicity to workers or consumers.
Justification for selection of repeated dose toxicity via oral
route - systemic effects endpoint:
Key study selected as most relevant study of highest concern.
Justification for selection of repeated dose toxicity inhalation -
systemic effects endpoint:
The inhalation study was waived because exposure is too low to exert
any toxicity. Strict exposure controls are based on the more sensitive
health effect of respiratory sensitization. Potential exposure by
inhalation to an amount of enzyme that is toxicologically relevant is
unrealistic due to the stringent work practices and the formulation of
enzymes.
Justification for selection of repeated dose toxicity inhalation -
local effects endpoint:
The inhalation study was waived because exposure is too low to exert
any toxicity. Strict exposure controls are based on the more sensitive
health effect of respiratory sensitization. Potential exposure by
inhalation to an amount of enzyme that is toxicologically relevant is
unrealistic due to the stringent work practices and the formulation of
enzymes.
Justification for selection of repeated dose toxicity dermal -
systemic effects endpoint:
Skin irritation was not seen in any of the test animals in the
single-dose study in rabbits. No signs of toxicity were noted. Repeated
dosing was not performed and is unlikely to produce any systemic effects
given the structure and properties of the enzyme protein. The overall
conclusion that pullulanase is non-toxic upon repeated dermal exposure
is supported by the knowledge of low possibility of absorption of
enzymes through the skin due to the physico-chemical properties of the
enzyme protein.
Justification for selection of repeated dose toxicity dermal - local
effects endpoint:
Skin irritation was not seen in any of the test animals in the
single-dose study in rabbits. No signs of toxicity were noted. Repeated
dosing was not performed and is unlikely to produce any systemic effects
given the structure and properties of the enzyme protein. The overall
conclusion that pullulanase is non-toxic upon repeated dermal exposure
is supported by the knowledge of low possibility of absorption of
enzymes through the skin due to the physico-chemical properties of the
enzyme protein.
Justification for classification or non-classification
Based on repeated dose oral study and weight of evidence, pullulanase does not exert any repeated dose oral, dermal or inhalation toxicity to workers or consumers.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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