Registration Dossier
Registration Dossier
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 232-752-2 | CAS number: 9014-01-1
- 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 subtilisin has been tested in a 90-day study, the repeated dose dermal toxicity was tested in a 28-day study, while the repeated dose inhalation toxicity was waived.
- The repeated dose oral toxicity was a subchronic toxicity test similar to the principles of OECD guideline 408 (adopted 1981), and in compliance with GLP. The conclusion was that the No Observed Adverse Effect Level (NOAEL) in rats was considered to be the intermediate dose level administered, equivalent to 400 mg of the subtilisin batch/kg bw/day or 360 mg Total Organic Solids (TOS)/kg bw/day.
- The dermal study concluded that the dose applied daily, 10 mg/kg/day (only one dose level tested), was without any significant effects, only sporadic, minimal grade local skin reactions. It is further highly unlikely that enzymes should be absorbed through the skin due to the molecular weight and the physico-chemical properties of the protein molecule.
- 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.
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:
- 09 February - 19 May, 1981
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Version / remarks:
- 1981
- Deviations:
- no
- Principles of method if other than guideline:
- Guideline OECD 408 was adopted for the first time in 1981, at the time where the present study was performed. The principles of the new guideline was however followed. The current version of OECD 408, revised in 1998, places additional emphasis on neurotoxic, immunological and reproductive organ effects as well as the need for more careful clinical observations of the animals compared to the 1981 version. These changes are not considered to be important for toxicological evaluation of enzymes produced by non-toxigenic, non-pathogenic strains for which the main safety issue of concern is respiratory allergy and in some cases primary irritation. This is supported by numerous 13-week oral toxicity studies performed on enzymes according to OECD 408 (1998), in which no effects on the additional parameters have been reported.
Therefore the present study is considered suitable for the evaluation of sub chronic toxicity of subtilisin. - 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
- Weight at study initiation: 60-80 g
- Fasting period before study: None
- Housing: 3 animals per cage, separate sex
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: 13 days
ENVIRONMENTAL CONDITIONS
- Temperature : 19-23°C
- Humidity : 50% ( range 26-80 %)
- Air changes (per hr): 14
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 1981-02-16 To: 1981-05-19 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- Distilled water
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 16, 40 and 100 mg/mL, corresponding to 14, 36 and 89 mg TOS/mL
- Amount of vehicle (if gavage): constant volume 10 mL/kg b.w.
- Purity: distilled water - Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- Daily
- Remarks:
- Doses / Concentrations:
0, 16, 40 and 100 mg/mL, corresponding to 0, 14, 36 and 89 mg TOS/mL
Basis:
nominal in water - No. of animals per sex per dose:
- 15
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Based on experience from dose-range studies, the top dose was selected to provide some toxicological effects but still avoiding death as an endpoint
- Positive control:
- Not included
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: early morning and late afternoon each day
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily and further more detailed examination of each animal once a week
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
FOOD CONSUMPTION:
- Food consumption for each cage, i.e. sum of three animals, was determined and mean daily diet consumption per group calculated as g feed/rat/week: Yes
WATER CONSUMPTION : Yes but only by visual assessment of water intake
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 6 and week 13
- Anaesthetic used for blood collection: Yes (ether anaesthesia)
- Animals fasted: No
- How many animals: 10 males and 10 females from negative control groups and high dose groups
- Parameters checked: Red blood cell count (RBC), White blood cell count (WBC), Packed cell volume (PCV), Haemoglobin conc (Hb), Differential leucocyte count, Platelet count.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 6 and week 13
- Animals fasted: No
- How many animals: 10 males and 10 females from negative control groups and high dose groups
- Parameters checked: Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, lactate dehydrogenase, blood urea nitrogen, Glucose, Sodium (Na), Potassium (K), Protein (total), Chloride (Cl), Cholesterol.
URINALYSIS: Yes
- Time schedule for collection of urine: Week 6 and week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Deprivation of feed and water during the 4-hour sampling period
- Parameters checked: Glucose, volume, protein, ketones, urobilinogen, colour, appearance, pH, specific gravity, microscopic examination of the spun deposit.
NEUROBEHAVIOURAL EXAMINATION: No data
The animals were observed daily in accordance with state of the art in 1981
OTHER: - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes.
The majority of the 16 premature decedents were found dead with red staining around nose and mouth. Most decedents showed red patches and oedema of the lungs. Three animals had haemorrhagic tracts or holes in the left lung lobe on post mortem examinination, consistent with lung damage as a result of gavage dosing. Both control and treated rats showed hyperplasia of the submandibular lymph nodes. This could be due to a previous sialodacryoadenitis virus infection. None of the lesions were attributed to toxic effects of the test material.
HISTOPATHOLOGY: Yes. Mild and focal alveolitis was identified in both control and treated animals - the cause was not identified. One animal showed a focal chronic alveolitis. Foreign material was identified in the alveolar air spaces of this animal, suggesting that the cause was inhalation or misdosing of foreign test material. - Other examinations:
- FAECAL ANALYSIS: Yes
- Time schedule for collection of faeces: Week 6 and week 13
- Metabolism cages used for collection: Yes
- Animals fasted: Deprivation of feed and water during the 4-hour sampling period
- Parameters presented in appendix 12, 13 and 14 were examined.
Weight of individual organs: yes
- Time schedule for collection of organs: At necropsy
- Results presented in table 12 and 13 . - Statistics:
- Organ weight and body weight data were statically analysed by computerized analysis of variance incorporating t-test. The level of probability chosen as significant was p<0.05.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- There were 16 premature deaths. Within males: 8 animals in the top dose group, 1 in mid dose group. Within female: Two animals in the top and mid groups, and 3 animals in the low dose group.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- There were 16 premature deaths. Within males: 8 animals in the top dose group, 1 in mid dose group. Within female: Two animals in the top and mid groups, and 3 animals in the low dose group.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Body weights were significantly reduced at the highest dose level in males.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- There was a slight dose-related reduction in food consumption in males. Food consumption in females was lower than controls but without dose-relation.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- There were no haematological changes considered to be related to treatment.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Urea nitrogen (BUN) and alanine aminotransferase (ALT) were significantly reduced at the highest dose level in males.
- Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- There were no abnormalities considered to be related to treatment.
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- The changes that were identified were not considered to of toxicological significance.
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- No lesions were seen at finalization of the study which could be attributed to toxic effects of the dosed test material. Many of the 16 premature deaths showed findings in the lungs from damage due to gavage dosing or unintended dosing of test material in
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- The changes that were identified were not considered of toxicological significance.
- Histopathological findings: neoplastic:
- not specified
- Details on results:
- CLINICAL SIGNS AND MORTALITY: The major clinical observation in this study was increased difficulty of dosing animals receiving 1000 mg/kg bw/day ~ 891 mg TOS/kg bw/day. This difficulty, resulting from struggling during dosing, caused many of the 16 premature deaths due to aspiration. When using oral gavage dosing, the dose volume of 10 ml/kg body weight can easily lead to unintended exposure by inhalation because test substance is deposited in the pharyngeal cavity after reflux from the stomach. This is a likely event in struggling animals, but is as such an artifact caused by the way of dosing the animals.
BODY WEIGHT AND WEIGHT GAIN: : Body weights were significantly reduced at the highest dose level in males.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
FOOD EFFICIENCY
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
OPHTHALMOSCOPIC EXAMINATION
HAEMATOLOGY: There were no haematological changes considered to be related to treatment.
CLINICAL CHEMISTRY: Clinical Urea nitrogen (BUN) and alanine aminotransferase (ALT) were significantly reduced at the highest dose level in males.
The reduced clinical chemical parameters BUN and ALT could be indicators of lever damage but due to lack of histopathological changes in the lever of these animals, this change is considered of no toxicological significance.
URINALYSIS
NEUROBEHAVIOUR
ORGAN WEIGHTS: The top dose males showed reduced absolute liver and spleen weights with no concurrent change in relative weights, unaltered absolute but increased relative brain weight, increased absolute and relative adrenal weights - could be the result of the difficulty in dosing (see reference 1 and 2 below). The males receiving 400 and 1000 mg/kg bw/day also showed increased relative lung weights, but this could be due to reduction in body weight.
References:
1. Scharer, K. (1977). The effect of the chronic underfeeding on organ weights of rats. Toxicology, 7: 45-46.
2. Oishi, S., Oishi H. and Hiraga, K. (1979). The effect of food restriction for 4 weeks on common toxicity parameters in male rats. Toxicol. Appl. Pharmacol., 47: 15-22.
The absolute and relative thyroid weights were found to be significantly increased in all treated males, but this finding is not judged to be of great significance for the following reasons:
1) The thyroid gland is small in rats with a color similar to the surrounding tissue and very difficult to dissect precisely for weighing. This is also indicated by the coefficient of variability (CV = 100 x SD/mean weight) which can be calculated from the thyroid weight data in table 12. The CV is above 20% in all groups and in the female control group, it is even 55%, showing a very high variability in these data.
2) The histopathology did not reveal any changes.
3) Historical data showed that the seen increase in weight was possibly due to unusual low thyroid weight data in the control animals for both males and females.
4) The present study was finalized Sept. 1981, and reports covering 13 week oral toxicity studies in rats from the same CRO in the following year, 1982 and onwards, does no longer include the thyroid weight as a parameter. This decision of excluding the organ weight of the thyroids is most likely taken because these data do not provide any added value. Further, the OECD test guideline 408, 'Subchronic Oral Toxicity - Rodent: 90-Day Study', which was adopted 12 May 1981, has supported this decision as weighing of the thyroid gland was not required in this guideline.
GROSS PATHOLOGY: The effects in the lungs (irregular brown areas) are considered artifacts originating from dosing by gavage especially in case of the higher dose level.
HISTOPATHOLOGY: NON-NEOPLASTIC : The effects in the lungs (brown pigments) are considered originating from dosing by gavage especially in case of the higher dose level.
HISTOPATHOLOGY: NEOPLASTIC (if applicable)
HISTORICAL CONTROL DATA (if applicable)
OTHER FINDINGS - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- > 360 - < 891 mg/kg bw/day (nominal)
- Based on:
- other: TOS
- Sex:
- male/female
- Basis for effect level:
- other: body weight: Slight reduction; food consumption: Slight reduction
- Critical effects observed:
- not specified
- Conclusions:
- The No Observed Adverse Effect Level (NOAEL) in rats is 400 mg of the subtilisin batch/kg bw/day equivalent to 360 mg Total Organic Solids (TOS)/kg bw/day.
- Executive summary:
Guideline OECD 408 was adopted for the first time in 1981, at the time where the present study was performed. The principles of this OECD guideline were, however, followed and the study performed in compliance with GLP. The current version of OECD 408, revised in 1998, places additional emphasis on neurotoxic, immunological and reproductive organ effects as well as the need for more careful clinical observations of the animals compared to the 1981 version. These changes are not considered to be important for toxicological evaluation of enzymes produced by non-toxigenic, non-pathogenic strains for which the main safety issue of concern is respiratory allergy and in some cases primary irritation. This is supported by several 13-week oral toxicity studies performed on enzymes according to OECD 408 (1998), in which no effects on the additional parameters have been reported. The present study is therefore considered suitable for the evaluation of sub chronic toxicity of subtilisin.
In conclusion, oral administration (by gavage) of subtilisin to rats at the highest dose level, 1000 mg subtilisin batch/kg bw/day, equivalent to 809 mg Total Organic Solids (TOS)/kg bw/dayfor thirteen weeks, resulted in significantly reduced body weights in males and remarkable struggling against the gavage. Apart from these effects, dosing did not produce any toxicologically significant changes. Consequently, the No Observed Adverse Effect Level (NOAEL) in rats was considered to be the intermediate dose level administered, equivalent to 400 mg of the subtilisin batch/kg bw/day or 360 mg Total Organic Solids (TOS)/kg bw/day.
Reference
For details, please see the attached report (scanned pdf file as it is written before electronic files were used).
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 360 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- High quality. The key study is GLP compliant and Klimisch 1.
Repeated dose toxicity: inhalation - systemic effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: inhalation
- Data waiving:
- exposure considerations
- 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:
- exposure considerations
- Justification for data waiving:
- other:
- Critical effects observed:
- not specified
Reference
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - systemic effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: dermal
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- other:
- Critical effects observed:
- not specified
- Endpoint:
- short-term repeated dose toxicity: dermal
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The subacute percutaneous toxicity of Subtilisin was assessed. The systemic and local effects of daily repeated applications of 10 mg/kg/day of the test material Subtilisin, prepared as a 0.5% w/v solution in water and 0.5% w/v in sodium tripolyphosphate buffer, respectively. The application was performed daily for 28 consecutive days without occlusion to the abraded and intact skin of the albino rabbit, an area equal to 10% of the total body surface clipped free of hair. Four female and four male rabbits were used per group, i.e. 32 rabbits in total including two negative control groups.
- GLP compliance:
- no
- Remarks:
- The study was performed before GLP was implemented but was performed according to state of the art at that time.
- Limit test:
- no
- Species:
- rabbit
- Strain:
- New Zealand White
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- - Source: Morton Commercial Rabbits, Parsonage Farm, Stanstedt, UK
- Fasting period before study: None
- Housing: individually in stainless steel cages
- Weight at study initiation: between 2.2 - 2.7 kg
- Age at study initiation: Young adults, 3 - 3.5 months
- Diet (e.g. ad libitum): Standard diet ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: minimum 14 days
- Temperature (°C): 14-18C
- Humidity : 50% ( range 40-60 %)
- Air changes (per hr): 17
- Photoperiod (hrs dark / hrs light): 10 hrs/14 hrs - Type of coverage:
- open
- Vehicle:
- other: water, respectively sodium tripolyphosphate buffer
- Details on exposure:
- TEST SITE
- Area of exposure: 10% of body surface
- Time intervals for shavings or clipplings: The animals were shaven as needed - no specific interval given in report.
REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: Four hours
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg/kg
- Concentration (if solution): 0.5% w/v
- Constant volume or concentration used: yes
- For solids, paste formed: no
VEHICLE
- Amount(s) applied (volume or weight with unit): 2 mL/kg
- Concentration (if solution): 0.1 % sodium tripolyphosphate buffer
USE OF RESTRAINERS FOR PREVENTING INGESTION: no - however, collars were worn by the animals four hours post-application. - Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- Four hours per day
- Frequency of treatment:
- Each day for 28 days.
- Remarks:
- Doses / Concentrations:
10 mg/kg, i.e. 2.0 mL/kg of a 0.5% w/v solution in water and sodium tripolyphosphate buffer, respectively.
Basis:
nominal per unit body weight - No. of animals per sex per dose:
- 4
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Based on experience from short term dose-range studies, the dose was selected to provide some toxicological effects but still avoiding extreme irritation as an endpoint
- Positive control:
- No positive control
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily
DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Days 1, 2, 3, 7, 14, 21, 28
BODY WEIGHT: Yes
- Time schedule for examinations: Weekly
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
WATER CONSUMPTION: No data
OPHTHALMOSCOPIC EXAMINATION: No data
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Before commencement and after 4 weeks of treatment
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 32
- Parameters checked in table No 4 & 5 were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Before commencement and after 4 weeks of treatment
- Animals fasted: No
- How many animals: 32
- Parameters checked in table No 6 & 7 were examined.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes ( Appendix 9 in report)
HISTOPATHOLOGY: Yes (Appendix 9 in report) - Other examinations:
- Twelve main organs were weighed
- Statistics:
- Organ weights were evaluated by analysis of variance.
- Clinical signs:
- no effects observed
- Dermal irritation:
- no effects observed
- Description (incidence and severity):
- skin reactions were confined to sporadic, minimal grade
- 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:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Any effects were confined to slight acanthosis, and a variable, slight infiltration of the underlying dermis by leucocytes
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- CLINICAL SIGNS AND MORTALITY: No clinical effects and no mortality
BODY WEIGHT AND WEIGHT GAIN: Within normal limits during the study.
FOOD CONSUMPTION: Within normal ranges during the study.
HAEMATOLOGY: Within normal ranges, unaffected by treatment.
CLINICAL CHEMISTRY Within normal ranges, unaffected by treatment.
NEUROBEHAVIOUR: Behaviour normal throughout the study
ORGAN WEIGHTS: No differences between groups.
GROSS PATHOLOGY: No treatment related gross lesions present
HISTOPATHOLOGY: NON-NEOPLASTIC There were no treatment related effects other than minor local skin effects. These were slight acanthosis, occasional patchy parakeratosis and variable slight infiltration of underlying dermis by leucocytes. - Key result
- Dose descriptor:
- conc. level:
- Effect level:
- > 10 mg/kg bw/day (nominal)
- Based on:
- dissolved
- Remarks:
- 0.5% w/v solution in water or buffer
- Sex:
- male/female
- Basis for effect level:
- other: detailed skin reactions, clinical signs; mortality; body weight; food consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology;
- Critical effects observed:
- not specified
- Conclusions:
- The only effects of 28-day repeated treatment of intact and abraded skin with Subtilisin, buffered and unbuffered, were microscopically detected minor changes at the site of application.
- Executive summary:
A percutaneous 28-day repeated application study in rabbits was conducted by Life Science Research (now Huntingdon Life Sciences Ltd.) to assess the potential of the test substance, Subtilisin (batch FPF 312/20), to cause dermal toxicity. Only one dose was applied daily, 10 mg/kg/day, diluted in either water or buffer, to the closely-clipped dorsa of New Zealand White rabbits, equal to 10% of the body surface. Vehicle controls were included. Each group consisted of 4 males and 4 females. The study was conducted before GLP was implemented but the principles was the same and state of the art was followed. The study concluded that the test substance, Subtilisin, was without any significant effects and thus may be considered virtually harmless.
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 10 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rabbit
- Quality of whole database:
- Klimisch 2 study - only one dose level tested.
Repeated dose toxicity: dermal - local effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: dermal
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- other:
- Critical effects observed:
- not specified
- Endpoint:
- short-term repeated dose toxicity: dermal
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The subacute percutaneous toxicity of Subtilisin was assessed. The systemic and local effects of daily repeated applications of 10 mg/kg/day of the test material Subtilisin, prepared as a 0.5% w/v solution in water and 0.5% w/v in sodium tripolyphosphate buffer, respectively. The application was performed daily for 28 consecutive days without occlusion to the abraded and intact skin of the albino rabbit, an area equal to 10% of the total body surface clipped free of hair. Four female and four male rabbits were used per group, i.e. 32 rabbits in total including two negative control groups.
- GLP compliance:
- no
- Remarks:
- The study was performed before GLP was implemented but was performed according to state of the art at that time.
- Limit test:
- no
- Species:
- rabbit
- Strain:
- New Zealand White
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- - Source: Morton Commercial Rabbits, Parsonage Farm, Stanstedt, UK
- Fasting period before study: None
- Housing: individually in stainless steel cages
- Weight at study initiation: between 2.2 - 2.7 kg
- Age at study initiation: Young adults, 3 - 3.5 months
- Diet (e.g. ad libitum): Standard diet ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: minimum 14 days
- Temperature (°C): 14-18C
- Humidity : 50% ( range 40-60 %)
- Air changes (per hr): 17
- Photoperiod (hrs dark / hrs light): 10 hrs/14 hrs - Type of coverage:
- open
- Vehicle:
- other: water, respectively sodium tripolyphosphate buffer
- Details on exposure:
- TEST SITE
- Area of exposure: 10% of body surface
- Time intervals for shavings or clipplings: The animals were shaven as needed - no specific interval given in report.
REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: Four hours
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg/kg
- Concentration (if solution): 0.5% w/v
- Constant volume or concentration used: yes
- For solids, paste formed: no
VEHICLE
- Amount(s) applied (volume or weight with unit): 2 mL/kg
- Concentration (if solution): 0.1 % sodium tripolyphosphate buffer
USE OF RESTRAINERS FOR PREVENTING INGESTION: no - however, collars were worn by the animals four hours post-application. - Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- Four hours per day
- Frequency of treatment:
- Each day for 28 days.
- Remarks:
- Doses / Concentrations:
10 mg/kg, i.e. 2.0 mL/kg of a 0.5% w/v solution in water and sodium tripolyphosphate buffer, respectively.
Basis:
nominal per unit body weight - No. of animals per sex per dose:
- 4
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Based on experience from short term dose-range studies, the dose was selected to provide some toxicological effects but still avoiding extreme irritation as an endpoint
- Positive control:
- No positive control
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily
DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Days 1, 2, 3, 7, 14, 21, 28
BODY WEIGHT: Yes
- Time schedule for examinations: Weekly
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
WATER CONSUMPTION: No data
OPHTHALMOSCOPIC EXAMINATION: No data
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Before commencement and after 4 weeks of treatment
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 32
- Parameters checked in table No 4 & 5 were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Before commencement and after 4 weeks of treatment
- Animals fasted: No
- How many animals: 32
- Parameters checked in table No 6 & 7 were examined.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes ( Appendix 9 in report)
HISTOPATHOLOGY: Yes (Appendix 9 in report) - Other examinations:
- Twelve main organs were weighed
- Statistics:
- Organ weights were evaluated by analysis of variance.
- Clinical signs:
- no effects observed
- Dermal irritation:
- no effects observed
- Description (incidence and severity):
- skin reactions were confined to sporadic, minimal grade
- 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:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Any effects were confined to slight acanthosis, and a variable, slight infiltration of the underlying dermis by leucocytes
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- CLINICAL SIGNS AND MORTALITY: No clinical effects and no mortality
BODY WEIGHT AND WEIGHT GAIN: Within normal limits during the study.
FOOD CONSUMPTION: Within normal ranges during the study.
HAEMATOLOGY: Within normal ranges, unaffected by treatment.
CLINICAL CHEMISTRY Within normal ranges, unaffected by treatment.
NEUROBEHAVIOUR: Behaviour normal throughout the study
ORGAN WEIGHTS: No differences between groups.
GROSS PATHOLOGY: No treatment related gross lesions present
HISTOPATHOLOGY: NON-NEOPLASTIC There were no treatment related effects other than minor local skin effects. These were slight acanthosis, occasional patchy parakeratosis and variable slight infiltration of underlying dermis by leucocytes. - Key result
- Dose descriptor:
- conc. level:
- Effect level:
- > 10 mg/kg bw/day (nominal)
- Based on:
- dissolved
- Remarks:
- 0.5% w/v solution in water or buffer
- Sex:
- male/female
- Basis for effect level:
- other: detailed skin reactions, clinical signs; mortality; body weight; food consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology;
- Critical effects observed:
- not specified
- Conclusions:
- The only effects of 28-day repeated treatment of intact and abraded skin with Subtilisin, buffered and unbuffered, were microscopically detected minor changes at the site of application.
- Executive summary:
A percutaneous 28-day repeated application study in rabbits was conducted by Life Science Research (now Huntingdon Life Sciences Ltd.) to assess the potential of the test substance, Subtilisin (batch FPF 312/20), to cause dermal toxicity. Only one dose was applied daily, 10 mg/kg/day, diluted in either water or buffer, to the closely-clipped dorsa of New Zealand White rabbits, equal to 10% of the body surface. Vehicle controls were included. Each group consisted of 4 males and 4 females. The study was conducted before GLP was implemented but the principles was the same and state of the art was followed. The study concluded that the test substance, Subtilisin, was without any significant effects and thus may be considered virtually harmless.
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 167 µg/cm²
- Study duration:
- subacute
- Species:
- rabbit
- Quality of whole database:
- Klimisch 2 study - only one dose level tested.
Additional information
The repeated dose oral toxicity of subtilisin has been tested in a 90-day study, the repeated dose dermal toxicity was tested in a 28-day study, while the repeated dose inhalation toxicity was waived.
- The repeated dose oral toxicity was a subchronic toxicity test similar to the principles of OECD guideline 408 (adopted 1981), and in compliance with GLP. The conclusion was that the No Observed Adverse Effect Level (NOAEL) in rats was considered to be the intermediate dose level administered, equivalent to 400 mg of the subtilisin batch/kg bw/day or 360 mg Total Organic Solids (TOS)/kg bw/day.
- The dermal study concluded that the dose applied daily, 10 mg/kg/day (only one dose tested), was without any significant effects, only sporadic, minimal grade local skin reactions. It is further highly unlikely that enzymes should be absorbed through the skin due to the molecular weight and the physico-chemical properties of the protein molecule.
- 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.
Therefore, based on weight of evidence, subtilisin does not exert any repeated dose inhalation toxicity to workers or consumers
Justification for selection of repeated dose toxicity via oral
route - systemic effects endpoint:
The present key study was selected based on quality, duration, GLP
status and lowest NOAEL.
Justification for selection of repeated dose toxicity inhalation -
systemic effects endpoint:
This endpoint study was waived as 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.
Justification for selection of repeated dose toxicity inhalation -
local effects endpoint:
This endpoint study was waived as 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.
Justification for selection of repeated dose toxicity dermal -
systemic effects endpoint:
The dermal study supports the information from the physico-chemical
properties of the enzyme molecule (high MW, logPow value <0), that
enzymes are not absorbed through the skin.
Justification for selection of repeated dose toxicity dermal - local
effects endpoint:
The only repeated dose study available.
Justification for classification or non-classification
No classification as NOAEL is above guidance level of 100 mg/kg bw/day. The installed controls to insure no risk of respiratory sensitization generally ensure no repeated dose inhalation or dermal 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.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

EU Privacy Disclaimer
This website uses cookies to ensure you get the best experience on our websites.