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EC number: 642-981-3 | CAS number: 148465-73-0
- 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
Short-term toxicity to fish
Administrative data
Link to relevant study record(s)
- Endpoint:
- short-term toxicity to fish
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 30 August - 24 November 2006
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Sublitisin read-across was employed as a worst-case scenario, due to it being a protease.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 203 (Fish, Acute Toxicity Test)
- Version / remarks:
- 1992
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- except for the analysis of the test material
- Analytical monitoring:
- yes
- Details on sampling:
- - Concentrations: Range finding test used concentrations 1.1, 10.7 and 106.8 mg enzyme concentrate dry matter/L. The definitive test concentrations were 6.7, 13.4, 26.7, 53.4 and 106.8 mg enzyme concentrate dry matter/L.
- Sampling method: Triplicate samples (10mL) of media were taken from the control and each test vessel at 0 and 72 hours (fresh media) and at 24 and 96 hours (expired media) for analysis.
- Sample storage conditions before analysis: Frozen - Vehicle:
- no
- Details on test solutions:
- PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:The test media were freshly prepared each day during the definitive test. As the test substance was supplied as a frozen liquid, an aliquot of the test substance was thawed on each occasions and discarded after use. At each concentration the test substance (1.54, 3.08, 6.16, 12.33 or 24.66 g) was dispersed in diluent water (5L). The contents of the flask were gently inverted thirty times to mix contents before being poured into test vessel (glass aquarium) and the volumes adjusted to 18 litres with diluent water.
- Controls: Medium - Test organisms (species):
- Oncorhynchus mykiss (previous name: Salmo gairdneri)
- Details on test organisms:
- TEST ORGANISM
- Common name: rainbow trout
- Strain: Oncorhoynchus mykiss
- Source: commercial fish farm in the UK
- Length at study initiation (length definition, mean): 5.84cm (4-6cm)
- Weight at study initiation (mean): 1.73g
- Feeding during test: no
ACCLIMATION
- Acclimation period: 11 days
- Acclimation conditions (same as test or not): the fish were gradually acclimated to the conditions of the study by gradual replacement of the holding tank water with pH adjusted diluent water. To maintain the levels of dissolved oxygen in the holding tank, the water was gently aerated, which resulted in an increase in pH levels between the additions of water. Each day during the seven day period before the definitive test, the pH of the holding water was returned to approximately 6.1-6.3 by the addition of pH adjusted diluent water.
- Type and amount of food: commercial fish food (TROUW (UK) Ltd., Nutra Fry 02); amount equivalent to 1% of the total wet-weight in the holding tank
- Feeding frequency: daily until 24 hours before exposure
- Health during acclimation (any mortality observed): no - Test type:
- semi-static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 96 h
- Hardness:
- 150-200 mg CaCO3/L
- Test temperature:
- 15 ± 2°C
- pH:
- 6.17-7.26
- Dissolved oxygen:
- 73-104 % air saturation value
- Conductivity:
- 330 µS/cm
- Nominal and measured concentrations:
- The nominal and measured concentrations for the definitive test were:
nominal: 6.7, 13.4, 26.7, 53.4 and 106.8 mg enzyme concentrate dry matter/L
measured: 3.4, 10.9, 22.4, 52.6, 142.1 mg enzyme concentrate dry matter/L - Details on test conditions:
- TEST SYSTEM
- Test vessel: glass aquarium
- Material, size, headspace, fill volume: glass, depth 17 cm, 18 litres of medium,
- Aeration: aeration provided via narrow bore glass tubes
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: initial static loading of 0.67 g body weight/litre
- Source/preparation of dilution water: The water used for the study was laboratory tap water filtered, dechlorinated and softened by passage through a Vivendi water purification system. The water was passed through a highgrade activated carbon filter to remove chlorine and any organic contaminants. A proportion of the supply was then passed through a water softener before final reverse osmosis (RO) treatment to produce a highly purified water supply. These two supplies were then remixed in the appropriate ratio to give a laboratory supply with the desired water hardness.
- Total organic carbon: 0.82 mg/L
- Chlorine: 0.05 mg/L (free chlorine 0.03 mg/L)
- Conductivity: 330 µS/cm
- Culture medium different from test medium: no
OTHER TEST CONDITIONS
- Adjustment of pH: to approximately 6-6.5
- Photoperiod: 16 hours light: 8 hours dark
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The criteria of death employed in this study were (i) absence of respiratory movement and (ii)absence of response to physical stimulation of the caudal peduncle. In addition to observations on mortality at approximately 2, 4, 24, 48, 72 and 96 hours, subjective assessments were made on the type and incidence of sub-lethal effects compared with control fish.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 1.1, 10.7 and 106.8 mg enzyme concentrate dry matter/L
- Results used to determine the conditions for the definitive study: Results from range finding study. - Reference substance (positive control):
- no
- Key result
- Duration:
- 96 h
- Dose descriptor:
- LC50
- Effect conc.:
- 15.6 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- other: mg enzyme concentrate dry matter/L
- Basis for effect:
- mortality (fish)
- Key result
- Duration:
- 96 h
- Dose descriptor:
- LC100
- Effect conc.:
- 52.6 mg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- other: mg enzyme concentrate dry matter/L
- Basis for effect:
- mortality (fish)
- Duration:
- 96 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 10.7 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- other: mg enzyme concentrate dry matter/L
- Basis for effect:
- mortality (fish)
- Details on results:
- - Behavioural abnormalities: During the initial four hours of exposure all fish including those in the control exhibited hyperventilation. This was attributed to the stringent test conditions employed in the test, with the fish taking longer to settle at the low pH after their addition to the vessel and therefor not considered treatment related. From 24 hours onwards, the fish in the control and test groups at 6.7 and 13.4 mg enzyme concentrate dry matter/L became acclimatised and from 48 hours onwards, all appeared normal
- Mortality of control: 0
- Abnormal responses: treatment related sub-lethal effects at 26.7 and 106.8 mg enzyme concentrate dry matter/L. - Reported statistics and error estimates:
- BERKSON,J. (1944) Application of logistic function to bio-assay. J.Amer. Statist.Assoc. 39,
357-365.
FISHER, R.A. (1973) Statistical Methods for Research Workers, 14th edn., p.96. Hafner
Publishing Company, New York, USA.
SAS INSTITUTE (1999) SAS OnlineDoc® Version Eight. SAS Institute Inc., Cary, NC, USA.
WILLIAMS,D.A. (1986) Interval Estimation of the median Lethal Dose. Biometrics, 42, 641-
645. - Validity criteria fulfilled:
- yes
- Conclusions:
- The measured 96-hour LC50 was 15.6 mg enzyme concentrate dry matter/L (measured as 8.2 mg active enzyme protein/L). Measured 100% mortality was 52.6 mg enzyme concentrate dry matter/L (measured as 27.6 mg active enzyme protein/L).
The NOEC was not clearly identified during the definitive test but based on the findings of a range finding test, it was 10.7 mg enzyme concentrate dry matter/L (nominal) (5.6 mg active enzyme protein/L). - Executive summary:
An acute toxicity test to rainbow trout (Oncorhynchus mykiss) was conducted to determine the effect of the test substance on fish. The study was conducted in accordance with OECD and EC guidelines, and in compliance with GLP. The analytical phase was not conducted according to GLP.
Groups of seven juvenile fish were exposed to subtilisin, dispersed in water at nominal concentrations of 6.7, 13.4, 26.7, 53.4 and 106.8 mg enzyme concentrate dry matter/L. The test media were prepared by direct addition of the test substance to diluent water; because the activity of the active component of the enzyme was reduced under alkaline conditions, the pH of the diluent water was adjusted to approximately 6.5 before use.
The test concentrations were verified based on the determination of the enzyme by ELISA. At the start of the test, the mean measured concentrations in samples of freshly prepared media ranged between 108 and 154% of their nominal values except at 6.7 mg enzyme concentrate dry matter/L, where the measured level was 65%. In the samples of freshly prepared media remaining at 72 hours (6.7 to 26.7 mg enzyme concentrate dry matter/L), the measured levels were lower, ranging between 45 and 72% of nominal. The reason for the difference between these two batches of media was not identified although an error during formulation of the test media was not supported by the raw data. In samples of expired (24-hours-old) media, the measured levels were substantially maintained (ranging between 77 and 115% of their starting values) except on one occasion at 13.4 mg enzyme concentrate dry matter/L where only 41%. Since the analytical results indicate that there was some variability in the exposure concentrations, the study results have been calculated in terms of both the nominal and mean measured concentrations.
Observations of the fish were made after approximately 2, 4, 24, 48, 72 and 96 hours of exposure. Based on these findings the following values have been estimated:
96-hour LC50: nominal 18.9 mg enzyme concentrate dry matter/L; measured 15.6 mg enzyme concentrate dry matter/L (measured as 8.2 mg active enzyme protein/L)
100% mortality: nominal 53.4 mg enzyme concentrate dry matter/L; measured 52.6 mg enzyme concentrate dry matter/L (measured as 27.6 mg active enzyme protein/L).
≤10% mortality: nominal 13.4 mg enzyme concentrate dry matter/L; measured 10.9 mg enzyme concentrate dry matter/L (measured as 5.7 mg active enzyme protein/L).
The no-observed-effect concentration (NOEC) was not clearly identified during the definitive test but based on the findings of a range finding test, the NOEC was 10.7 mg enzyme concentrate dry matter/L (nominal) (5.6 mg active enzyme protein/L).
Reference
Description of key information
The measured 96-hour LC50 was 15.6 mg enzyme concentrate dry matter/L (measured as 8.2 mg active enzyme protein/L). Measured 100% mortality was 52.6 mg enzyme concentrate dry matter/L (measured as 27.6 mg active enzyme protein/L).
The NOEC was not clearly identified during the definitive test but based on the findings of a range finding test, it was 10.7 mg enzyme concentrate dry matter/L (nominal) (5.6 mg active enzyme protein/L).
Key value for chemical safety assessment
Fresh water fish
Fresh water fish
- Dose descriptor:
- EC50
- Effect concentration:
- 15.6 mg/L
Additional information
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