Inhaltsverzeichnis Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance) (Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursu... (32008R0440))

Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursu... (32008R0440)

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Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)

COUNCIL REGULATION (EC)
No 440/2008
of 30 May 2008
laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
(Text with EEA relevance)
Article 1
Article 2
Article 3
Article 4
ANNEX
PART A: METHODS FOR THE DETERMINATION OF PHYSICO-CHEMICAL PROPERTIES
A.1. MELTING/FREEZING TEMPERATURE
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.4.1. Capillary method
1.4.1.1. Melting temperature devices with liquid bath
1.4.1.2. Melting temperature devices with metal block
1.4.1.3. Photocell detection
1.4.2. Hot stages
1.4.2.1. Kofler hot bar
1.4.2.2. Melt microscope
1.4.2.3. Meniscus method
1.4.3. Method to determine the freezing temperature
1.4.4. Thermal analysis
1.4.4.1 Differential thermal analysis (DTA)
1.4.4.2 Differential scanning calorimetry (DSC)
1.4.5. Pour point
1.5. QUALITY CRITERIA
TABLE: APPLICABILITY OF THE METHODS
A.
Capillary methods
B.
Hot stages and freezing methods
C.
Thermal analysis
D.
Pour point
1.6. DESCRIPTION OF THE METHODS
1.6.1. Methods with capillary tube
Figure 1
1.6.1.1. Melting temperature devices with liquid bath apparatus
Figure 2
Bath liquid:
Thermometer:
Procedure:
Calculation:
1.6.1.2. Melting temperature devices with metal block
Apparatus:
Thermometer:
Figure 3
1.6.1.3. Photocell detection
1.6.2. Hot stages
1.6.2.1. Kofler hot bar
1.6.2.2. Melt microscope
1.6.2.3. Meniscus method (polyamides)
1.6.3. Methods for the determination of the freezing temperature
1.6.4. Thermal analysis
1.6.4.1. Differential thermal analysis
1.6.4.2. Differential scanning calorimetry
1.6.5. Determination of the pour point
2. DATA
3. REPORTING
4. REFERENCES
Appendix
For additional technical details, the following standards may be consulted for example.
1. Capillary methods
1.1. Melting temperature devices with liquid bath
1.2. Melting temperature devices with metal block
2. Hot stages
2.1. Kofler hot bar
2.2. Melt microscope
2.3. Meniscus method (polyamides)
3. Methods for the determination of the freezing temperature
4. Thermal analysis
4.1. Differential thermal analysis
4.2. Differential scanning calorimetry
5. Determination of the pour point
A.2. BOILING TEMPERATURE
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.4.1. Determination by use of the ebulliometer
1.4.2. Dynamic method
1.4.3. Distillation method for boiling temperature
1.4.4. Method according to Siwoloboff
1.4.5. Photocell detection
1.4.6. Differential thermal analysis
1.4.7. Differential scanning calorimetry
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE METHODS
1.6.1. Ebulliometer
1.6.2. Dynamic method
1.6.3. Distillation process (boiling range)
1.6.4. Method according to Siwoloboff
Figure 1
1.6.5. Photocell detection
1.6.6. Thermal analysis
1.6.6.1. Differential thermal analysis
1.6.6.2. Differential scanning calorimetry
2. DATA
3. REPORTING
4. REFERENCES
Appendix
For additional technical details, the following standards may be consulted for example.
1. Ebulliometer
2. Distillation process (boiling range)
3. Differential thermal analysis and differential scanning calorimetry
A.3. RELATIVE DENSITY
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES (1) (3)
1.4. PRINCIPLE OF THE METHODS
1.4.1. Buoyancy methods
1.4.1.1. Hydrometer
(for liquid substances)
1.4.1.2. Hydrostatic balance
(for liquid and solid substances)
1.4.1.3. Immersed body method
(for liquid substances) (4)
1.4.2. Pycnometer methods
1.4.3. Air comparison pycnometer
(for solids)
1.4.4. Oscillating densitimeter
(5) (6) (7)
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE METHODS
2. DATA
3. REPORTING
4. REFERENCES
Appendix
For additional technical details, the following standards may be consulted for example.
1. Buoyancy methods
1.1. Hydrometer
1.2. Hydrostatic balance
For solid substances
For liquid substances
1.3. Immersed body method
2. Pycnometer methods
2.1. For liquid substances
2.2. For solid substances
3. Air comparison pycnometer
A.4. VAPOUR PRESSURE
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHODS
1.4.1. Dynamic method
1.4.2. Static method
1.4.3. Isoteniscope
1.4.4. Effusion method: Vapour pressure balance
1.4.5. Effusion method: By loss of weight or by trapping vaporisate
1.4.6. Gas saturation method
1.4.7. Spinning rotor
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE METHODS
1.6.1. Dynamic measurement
1.6.1.1. Apparatus
1.6.1.2. Measurement procedure
1.6.2. Static measurement
1.6.2.1. Apparatus
1.6.2.2. Measurement procedure
1.6.3. Isoteniscope
1.6.4. Effusion method: vapour pressure balance
1.6.4.1. Apparatus
1.6.4.2. Measurement procedure
1.6.5. Effusion method — by loss of weight
1.6.5.1. Apparatus
1.6.5.2. Measurement procedure
1.6.6. Gas saturation method
1.6.6.1. Apparatus
Inert gas:
Flow control:
Traps to collect vapour:
Heat exchanger:
Saturator column:
1.6.6.2. Measurement procedure
Preparation of the saturator column:
Measurement:
1.6.6.3. Calculation of vapour pressure
1.6.7. Spinning rotor (8, 11, 13)
1.6.7.1. Apparatus
1.6.7.2. Measurement procedure
2. DATA
3. REPORTING
4. REFERENCES
Appendix 1
Estimation method
INTRODUCTION
ESTIMATION METHOD
CALCULATION PROCEDURE
REPORT
LITERATURE
Appendix 2
Figure 1
Apparatus for determining the vapour pressure curve according to the dynamic method
Figure 2a
Apparatus for determining the vapour pressure curve according to the static method (using a U-tube manometer)
Figure 2b
Apparatus for determining the vapour pressure curve according to the static method (using a pressure indicator)
Figure 3
Isoteniscope (see reference 7)
Figure 4
Apparatus for determining the vapour pressure curve according to the vapour pressure balance method
Figure 5
Example of apparatus for evaporation at low pressure by effusion methode, with an effusion cell volume of 8 cm
3
Figure 6a
An example of a flow system for the determination of vapour pressure by the gas saturation method
Figure 6b
An example of system for the determination of vapour pressure by the gas saturation method, with a capillary placed after the saturation chamber
Figure 7
Example of the experimental set-up for spinning rotor method
Figure 8
Example of spinning rotor measuring head
A.5. SURFACE TENSION
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE METHODS
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE METHODS
1.6.1. Plate method
1.6.2. Stirrup method
1.6.3. Ring method
1.6.4. OECD harmonised ring method
1.6.4.1. Apparatus
1.6.4.1.1.
Mobile sample table
1.6.4.1.2.
Force measuring system
1.6.4.1.3.
Measuring body (ring)
Figure
Measuring body
1.6.4.1.4.
Measurement vessel
1.6.4.2. Preparation of the apparatus
1.6.4.2.1.
Cleaning
1.6.4.2.2.
Calibration of the apparatus
Mounting:
Zero point adjustment:
Calibrations:
1.6.4.3. Preparation of samples
1.6.5. Test conditions
1.6.6. Performance of test
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. INTERPRETATION OF RESULTS
4. REFERENCES
A.6. WATER SOLUBILITY
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.4.1. Column elution method
1.4.2. Flask method
1.5. QUALITY CRITERIA
1.5.1. Repeatability
1.5.2. Sensitivity
1.6. DESCRIPTION OF THE METHOD
1.6.1. Test conditions
1.6.2. Preliminary test
1.6.3. Column elution method
1.6.3.1. Support material, solvent and eluent
1.6.3.2. Loading of the support
Test procedure:
1.6.3.3. Column elution method with recirculating pump
Apparatus
Measurement procedure
1.6.3.4. Column elution method with levelling vessel
Apparatus
(see figures 4 and 3)
Measurement procedure
1.6.4. Flask method
1.6.4.1. Apparatus
1.6.4.2. Measurement procedure
1.6.5. Analysis
2. DATA
2.1. COLUMN ELUTION METHOD
2.2. FLASK METHOD
3. REPORTING
3.1. COLUMN ELUTION METHOD
3.2. FLASK METHOD
4. REFERENCES
Appendix
Figure 1
Column elution method with recirculating pump
Figure 2
A typical microcolumn
Figure 3
A typical microcolumn
Figure 4
Column elution method with levelling vessel
A.8. PARTITION COEFFICIENT
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION AND UNITS
1.3. REFERENCE SUBSTANCES
Shake-flask method
HPLC method
1.4. PRINCIPLE OF THE METHOD
1.4.1. Shake-flask method
1.4.2. HPLC method
1.5. QUALITY CRITERIA
1.5.1. Repeatability
Shake-flask method
HPLC method
1.5.2. Sensitivity
Shake-flask method
HPLC method
1.5.3. Specificity
Shake-flask method
1.6. DESCRIPTION OF THE METHOD
1.6.1. Preliminary estimate of the partition coefficient
1.6.2. Shake-flask method
1.6.2.1. Preparation
1.6.2.1.1.
Pre-saturation of the solvents
1.6.2.1.2.
Preparation for the test
1.6.2.1.3.
Test substance
1.6.2.2. Test conditions
1.6.2.3. Measurement procedure
1.6.2.3.1.
Establishment of the partition equilibrium
1.6.2.3.2.
Phase separation
1.6.2.4. Analysis
1.6.3. HPLC method
1.6.3.1. Preparation
Apparatus
Mobile phase
Solutes
Test conditions
1.6.3.2.
Measurement
Calculation of dead time t
o
Calibration graph
Determination of the capacity factor of the test substance
2. DATA
Shake-flask method
3. REPORTING
For shake-flask method:
For HPLC method:
4. REFERENCES
Appendix 1
Calculation/estimation methods
INTRODUCTION
ESTIMATION METHOD
Preliminary estimate of the partition coefficient
CALCULATION METHODS
Principle of the calculation methods
Quality criteria
Calculation procedures
Hansch π-method
Rekker method
Hansch-Leo method
Combined method
Remarks
Report
LITERATURE
Appendix 2
Recommended Reference Substances for the HLPC Method
A.9. FLASH-POINT
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE METHOD
1.5. QUALITY CRITERIA
1.5.1. Repeatability
1.5.2. Sensitivity
1.5.3. Specificity
1.6. DESCRIPTION OF THE METHOD
1.6.1. Preparations
1.6.2. Test conditions
1.6.3. Performance of the test
1.6.3.1. Equilibrium method
1.6.3.2. Non-equilibrium method
Abel apparatus:
Abel-Pensky apparatus:
Tag apparatus:
Pensky-Martens apparatus:
Remarks:
3. REPORTING
4. REFERENCES
A.10. FLAMMABILITY (SOLIDS)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF METHOD
1.6.1. Preliminary screening test
1.6.2. Burning rate test
1.6.2.1. Preparation
1.6.2.2. Test conditions
1.6.2.3. Performance of the test
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. INTERPRETATION OF THE RESULT
4. REFERENCES
Appendix
Figure
Mould and accessories for the preparation of the pile
A.11. FLAMMABILITY (GASES)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE METHOD
1.6.1. Apparatus
1.6.2. Test conditions
1.6.3. Performance of the test
2. DATA
3. REPORTING
4. REFERENCES
A.12. FLAMMABILITY (CONTACT WITH WATER)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. PRINCIPLE OF THE METHOD
1.3.1. Step 1
1.3.2. Step 2
1.3.3. Step 3
1.3.4. Step 4
1.4. REFERENCE SUBSTANCES
1.5. QUALITY CR1TERIA
1.6. DESCRIPTION OF METHODS
1.6.1. Step 1
1.6.1.1. Test conditions
1.6.1.2. Performance of the test
1.6.2. Step 2
1.6.2.1. Apparatus
1.6.2.2. Test conditions
1.6.2.3. Performance of the test
1.6.3. Step 3
1.6.3.1. Test conditions
1.6.3.2. Performance of the test
1.6.4. Step 4
1.6.4.1. Apparatus
1.6.4.2. Test conditions
1.6.4.3. Performance of the test
2. DATA
3. REPORTING
4. REFERENCES
Appendix
Figure
Apparatus
A.13. PYROPHORIC PROPERTIES OF SOLIDS AND LIQUIDS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Apparatus
1.6.2. Performance of the test
(a) Powdery solids
(b) liquids
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION
3. REPORTING
4. REFERENCES
A.14. EXPLOSIVE PROPERTIES
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE METHOD
1.4.1. Safety-in-handling tests (3)
1.4.2. Thermal sensitivity
1.4.3. Mechanical sensitivity (shock)
1.4.4. Mechanical sensitivity (friction)
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF METHOD
1.6.1. Thermal sensitivity (effect of a flame)
1.6.1.1. Apparatus
1.6.1.2. Test conditions
1.6.1.3. Performance of the tests
1.6.1.4. Evaluation
1.6.2. Mechanical sensitivity (shock)
1.6.2.1. Apparatus (figure 4)
1.6.2.2. Test conditions
1.6.2.3. Performance of the tests
1.6.2.4. Evaluation
1.6.3. Mechanical sensitivity (friction)
1.6.3.1. Apparatus (figure 5)
1.6.3.2. Test conditions
1.6.3.3. Performance of the tests
1.6.3.4. Evaluation
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. INTERPRETATION AND EVALUATION OF RESULTS
4. REFERENCES
Appendix
Example of material specification for thermal sensitivity test (see DIN 1623)
Figure 1
Thermal sensitivity test apparatus
Figure 2
Thermal sensitivity test
Figure 3
Heating rate calibration for thermal sensitivity test
Figure 4
Shock test apparatus
Figure 4
Continued
Figure 5
Friction sensitivity apparatus
A.15. AUTO-IGNITION TEMPERATURE (LIQUIDS AND GASES)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE METHOD
1.6.1. Apparatus
1.6.2. Test conditions
1.6.3. Performance of the test
2. DATA
3. REPORTING
4. REFERENCES
A.16. RELATIVE SELF-IGNITION TEMPERATURE FOR SOLIDS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCE
1.4. PRINCIPLE OF THE METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE METHOD
1.6.1. Apparatus
1.6.1.1. Oven
1.6.1.2. Wire mesh cube
1.6.1.3. Thermocouples
1.6.1.4. Recorder
1.6.2. Test conditions
1.6.3. Performance of the test
2. DATA
3. REPORTING
4. REFERENCES
Figure 1
Pattern of 20 mm test cube
Figure 2
Typical temperature/time curve
A.17. OXIDISING PROPERTIES (SOLIDS)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION AND UNITS
1.3. REFERENCE SUBSTANCE
1.4. PRINCIPLE OF THE METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE METHOD
1.6.1. Preparation
1.6.1.1. Test substance
1.6.1.2. Combustible substance
1.6.1.3. Ignition source
1.6.2. Performance of the test
1.6.2.1. Preliminary test
1.6.2.2. Train test
2. DATA
3. REPORT
3.1. TEST REPORT
3.2. INTERPRETATION OF THE RESULT
4. REFERENCES
Appendix
Figure
Mould and accessories for the preparations of the pile
A.18. NUMBER-AVERAGE MOLECULAR WEIGHT AND MOLECULAR WEIGHT DISTRIBUTION OF POLYMERS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Preparation of the standard polystyrene solutions
1.6.2. Preparation of the sample solution
1.6.3. Apparatus
1.6.4. Injection and solvent delivery system
1.6.5. Column
1.6.6. Theoretical plates
1.6.7. Separation efficiency
1.6.8. Solvents
1.6.9. Temperature control
1.6.10. Detector
2. DATA AND REPORTING
2.1. DATA
2.2. TEST REPORT
2.2.1. Test substance:
2.2.2. Instrumentation:
2.2.3. Calibration of the system:
2.2.4. Evaluation:
3. REFERENCES
Appendix
Examples of other methods for determination of number average molecular weight (Mn) for polymers
1. Use of colligative properties
2. End-group analysis
3. References
A.19. LOW MOLECULAR WEIGHT CONTENT OF POLYMERS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Preparation of the standard polystyrene solutions
1.6.2. Preparation of the sample solution
1.6.3. Correction for content of impurities and additives
1.6.4. Apparatus
1.6.5. Injection and solvent delivery system
1.6.6. Column
1.6.7. Theoretical plates
1.6.8. Separation efficiency
1.6.9. Solvents
1.6.10. Temperature control
1.6.11. Detector
2. DATA AND REPORTING
2.1. DATA
2.2. TEST REPORT
2.2.1. Test substance:
2.2.2. Instrumentation:
2.2.3. Calibration of the system:
2.2.4. Information on the low molecular weight polymer content:
2.2.5. Evaluation:
3. REFERENCES
Appendix
Guidance for correcting low molecular content for the presence of insoluble polymer
A.20. SOLUTION/EXTRACTION BEHAVIOUR OF POLYMERS IN WATER
1. METHOD
1.1. INTRODUCTION
1.2. REFERENCE SUBSTANCES
1.3. PRINCIPLE OF THE TEST METHOD
1.4. QUALITY CRITERIA
1.5. DESCRIPTION OF THE TEST METHOD
1.5.1. Equipment
1.5.2. Sample preparation
1.5.3. Procedure
1.5.4. Analysis
1.5.4.1. Test conducted with one sample size
1.5.4.2. Test conducted with two different sample sizes
2. DATA
2.1. TEST CONDUCTED WITH ONE SAMPLE SIZE
2.2. TEST CONDUCTED WITH TWO DIFFERENT SAMPLE SIZES
3. REPORTING
3.1. TEST REPORT
3.1.1. Test substance:
3.1.2. Experimental conditions:
3.1.3. Results:
4. REFERENCES
A.21. OXIDISING PROPERTIES (LIQUIDS)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCE
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE METHOD
1.6.1. Preparation
1.6.1.1. Combustible substance
1.6.1.2. Apparatus
1.6.1.2.1.
Pressure vessel
1.6.1.2.2.
Ignition system
1.6.2. Performance of the test
(15)
2. DATA
2.1. TREATMENT OF RESULTS
3. REPORT
3.1. TEST REPORT
3.2. INTERPRETATION OF THE RESULTS(22)
4. REFERENCES
Figure 1
Pressure vessel
Figure 2
Support stand
Figure 3
Ignition system
PART B: METHODS FOR THE DETERMINATION OF TOXICITY AND OTHER HEALTH EFFECTS
GENERAL INTRODUCTION
A. CHARACTERISATION OF THE TEST SUBSTANCE
B. ANIMAL CARE
(i) Housing conditions
(ii) Feeding conditions
C. ALTERNATIVE TESTING
D. EVALUATION AND INTERPRETATION
E. LITERATURE REFERENCES
B.1 bis. ACUTE ORAL TOXICITY — FIXED DOSE PROCEDURE
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Selection of animal species
1.4.2. Housing and feeding conditions
1.4.3. Preparation of animals
1.4.4. Preparation of doses
1.5. PROCEDURE
1.5.1. Administration of doses
1.5.2. Sighting study
1.5.3. Main study
1.5.3.1. Numbers of animals and dose levels
1.5.3.2. Limit test
1.6. OBSERVATIONS
1.6.1. Body weight
1.6.2. Pathology
2. DATA
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Appendix 1
FLOW CHART FOR THE SIGHTING STUDY
Appendix 2
FLOW CHART FOR THE MAIN STUDY
Appendix 3
CRITERIA FOR CLASSIFICATION OF TEST SUBSTANCES WITH EXPECTED LD
50
VALUES EXCEEDING 2 000 MG/KG WITHOUT THE NEED FOR TESTING.
TESTING AT DOSES ABOVE 2 000 MG/KG
Sighting study
Main study
Appendix 4
TEST METHOD B.1 bis
Guidance on classification according to the EU scheme to cover the transition period until full implementation of the Globally Harmonised Classification System (GHS) (taken from reference (8))
B.1 tris. ACUTE ORAL TOXICITY — ACUTE TOXIC CLASS METHOD
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST
1.4. DESCRIPTION OF THE METHOD
1.4.1. Selection of animal species
1.4.2. Housing and feeding conditions
1.4.3. Preparation of animals
1.4.4. Preparation of doses
1.5. PROCEDURE
1.5.1. Administration of doses
1.5.2. Number of animals and dose levels
1.5.3. Limit test
1.6. OBSERVATIONS
1.6.1. Body weight
1.6.2. Pathology
2. DATA
3. REPORTING
3.1. Test report
4. REFERENCES
Appendix 1
PROCEDURE TO BE FOLLOWED FOR EACH OF THE STARTING DOSES
GENERAL REMARKS
Appendix 1A
TEST PROCEDURE WITH A STARTING DOSE OF 5 MG/KG BODY WEIGHT
Appendix 1B
TEST PROCEDURE WITH A STARTING DOSE OF 50 MG/KG BODY WEIGHT
Appendix1C
TEST PROCEDURE WITH A STARTING DOSE OF 300 MG/KG BODY WEIGHT
Appendix 1D
TEST PROCEDURE WITH A STARTING DOSE OF 2 000 MG/KG BODY WEIGHT
Appendix 2
CRITERIA FOR CLASSIFICATION OF TEST SUBSTANCES WITH EXPECTED LD
50
VALUES EXCEEDING 2 000 MG/KG WITHOUT THE NEED FOR TESTING
TESTING AT DOSES ABOVE 2 000 MG/KG
Appendix 3
TEST METHOD B.1 tris: Guidance on classififcation according to EU scheme to cover the transition period until full implementation of the Globally Harmonised Classification System (GHS) (taken from reference (8))
B.2. ACUTE TOXICITY (INHALATION)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Preparations
1.6.2. Test conditions
1.6.2.1. Experimental animals
1.6.2.2. Number and sex
1.6.2.3. Exposure concentrations
1.6.2.4. Limit test
1.6.2.5. Exposure time
1.6.2.6. Equipment
1.6.2.7. Observation period
1.6.3. Procedure
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.3. ACUTE TOXICITY (DERMAL)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Preparations
1.6.2. Test conditions
1.6.2.1. Experimental animals
1.6.2.2. Number and sex
1.6.2.3. Dose levels
1.6.2.4. Limit test
1.6.2.5. Observation period
1.6.3. Procedure
Assessment of toxicity in the other sex
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.4. ACUTE TOXICITY: DERMAL IRRITATION/CORROSION
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparation for the
in vivo
test
1.4.1.1. Selection of animal species
1.4.1.2. Preparation of the animals
1.4.1.3. Housing and feeding conditions
1.4.2. Test procedure
1.4.2.1. Application of the test substance
1.4.2.2. Dose level
1.4.2.3. Initial test (
in vivo
dermal irritation/corrosion test using one animal)
1.4.2.4. Confirmatory test (
in vivo
dermal irritation test with additional animals)
1.4.2.5. Observation period
1.4.2.6. Clinical observations and grading of skin reactions
2. DATA
2.1. PRESENTATION OF RESULTS
2.2. EVALUATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Table I
GRADING OF SKIN REACTIONS
Appendix
A Sequential Testing Strategy for Dermal Irritation and Corrosion
GENERAL CONSIDERATIONS
DESCRIPTION OF THE EVALUATION AND TESTING STRATEGY
REFERENCES
Figure
TESTING AND EVALUATION STRATEGY FOR DERMAL IRRITATION/CORROSION
B.5. ACUTE TOXICITY: EYE IRRITATION/CORROSION
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparation for the
in vivo
test
1.4.1.1. Selection of species
1.4.1.2. Preparation of animals
1.4.1.3. Housing and feeding conditions
1.4.2. Test procedure
1.4.2.1. Application of the test substance
1.4.2.2. Irrigation
1.4.2.3. Dose level
1.4.2.3.1.
Testing of liquids
1.4.2.3.2.
Testing of solids
1.4.2.3.3.
Testing of aerosols
1.4.2.4. Initial test (
in vivo
eye irritation/corrosion test using one animal)
1.4.2.5. Local anaesthetics
1.4.2.6. Confirmatory test (
in vivo
eye irritation test with additional animals)
1.4.2.7. Observation period
1.4.2.7.1.
Clinical observations and grading of eye reactions
2. DATA
2.2. EVALUATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
3.2. INTERPRETATION OF THE RESULTS
4. REFERENCES
Table I
GRADING OF OCULAR LESIONS
Cornea
Iris
Conjunctivae
Chemosis
Appendix
A Sequential Testing Strategy for Eye Irritation and Corrosion
GENERAL CONSIDERATIONS
DESCRIPTION OF THE STEPWISE TESTING STRATEGY
REFERENCES
Figure
TESTING AND EVALUATION STRATEGY FOR EYE IRRITATION/CORROSION
B.6. SKIN SENSITISATION
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. DESCRIPTION OF THE TEST METHODS
1.5.1.
Guinea-Pig Maximisation Test (GPMT)
1.5.1.1.
Preparations
1.5.1.2.
Test conditions
1.5.1.2.1. Test animals
1.5.1.2.2. Number and sex
1.5.1.2.3. Dose levels
1.5.1.3.
Procedure
1.5.1.3.1. Induction
1.5.1.3.2. Challenge
1.5.1.3.3. Observation and Grading: treated and control groups
1.5.2.
Buehler test
1.5.2.1.
Preparations
1.5.2.2.
Test conditions
1.5.2.2.1. Test animals
1.5.2.2.2. Number and sex
1.5.2.2.3. Dose levels
1.5.2.3.
Procedure
1.5.2.3.1. Induction
1.5.2.3.2. Challenge
1.5.2.3.3. Observation and grading
2. DATA (GPMT and Buehler test)
3. REPORTING (GPMT and Buehler test)
Test report (GMPT and Buehler test)
4. REFERENCES
Appendix
TABLE
Magnusson/Kligman grading scale for the evaluation of challenge patch test reactions
B.7. REPEATED DOSE (28 DAYS) TOXICITY (ORAL)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations
1.4.2. Test conditions
1.4.2.1. Test animals
1.4.2.2. Number and sex
1.4.2.3. Dose levels
1.4.2.4. Limit test
1.4.2.5. Observation period
1.4.3. Procedure
1.4.3.1. General observations
1.4.3.2. Body weight and food/water consumption
1.4.3.3. Haematology
1.4.3.4. Clinical biochemistry
1.4.3.5. Gross necropsy
1.4.3.6. Histopathological examination
2. DATA
3. REPORTING
TEST REPORT
4. REFERENCES
B.8. REPEATED DOSE (28 DAYS) TOXICITY (INHALATION)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Preparations
1.6.2. Test conditions
1.6.2.1. Experimental animals
1.6.2.2. Number and sex
1.6.2.3. Exposure concentration
1.6.2.4. Exposure time
1.6.2.5. Equipment
1.6.2.6. Observation period
1.6.3. Procedure
1.6.3.1. Gross necropsy
1.6.3.2. Histopathological examination
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.9. REPEATED DOSE (28 DAYS) TOXICITY (DERMAL)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Preparations
1.6.2. Test conditions
1.6.2.1. Experimental animals
1.6.2.2. Number and sex
1.6.2.3. Dose levels
1.6.2.4. Limit test
1.6.2.5. Observation period
1.6.3. Procedure
1.6.4. Gross necropsy
1.6.5. Histopathological examination
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.10. MUTAGENICITY —
IN VITRO
MAMMALIAN CHROMOSOME ABERRATION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations
1.4.1.1. Cells
1.4.1.2. Media and culture conditions
1.4.1.3. Preparation of cultures
1.4.1.4. Metabolic activation
1.4.1.5. Test substance/Preparation
1.4.2. Test conditions
1.4.2.1. Solvent/vehicle
1.4.2.2. Exposure concentrations
1.4.2.3. Negative and positive controls
1.4.3. Procedure
1.4.3.1. Treatment with the test substance
1.4.3.3. Culture harvest time
1.4.3.4. Chromosome preparation
1.4.3.5. Analysis
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
TEST REPORT
4. REFERENCES
B.11. MUTAGENICITY —
IN VIVO
MAMMALIAN BONE MARROW CHROMOSOME ABERRATION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations
1.4.1.1. Selection of animal species
1.4.1.2. Housing and feeding conditions
1.4.1.3. Preparation of the animals
1.4.1.4. Preparation of doses
1.4.2. Test conditions
1.4.2.1. Solvent/Vehicle
1.4.2.2. Controls
1.5. PROCEDURE
1.5.1. Number and sex of animals
1.5.2. Treatment schedule
1.5.3. Dose levels
1.5.4. Limit test
1.5.5. Administration of doses
1.5.6. Chromosome preparation
1.5.7. Analysis
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
B.12. MUTAGENICITY —
IN VIVO
MAMMALIAN ERYTHROCYTE MICRONUCLEUS TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations
1.4.1.1. Selection of animal species
1.4.1.2. Housing and feeding conditions
1.4.1.3. Preparation of the animals
1.4.1.4. Preparation of doses
1.4.2. Test conditions
1.4.2.1. Solvent/Vehicle
1.4.2.2. Controls
1.5. PROCEDURE
1.5.1. Number and sex of animals
1.5.2. Treatment schedule
1.5.3. Dose levels
1.5.4. Limit test
1.5.5. Administration of doses
1.5.6. Bone marrow/blood preparation
1.5.7. Analysis
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
TEST REPORT
4. REFERENCES
B.13/14. MUTAGENICITY: REVERSE MUTATION TEST USING BACTERIA
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. INITIAL CONSIDERATIONS
1.4. PRINCIPLE OF THE TEST METHOD
1.5. DESCRIPTION OF THE TEST METHOD
1.5.1. Preparations
1.5.1.1. Bacteria
1.5.1.2. Medium
1.5.1.3. Metabolic activation
1.5.1.4. Test substance/Preparation
1.5.2. Test conditions
1.5.2.1. Test strains (see 1.5.1.1)
1.5.2.2. Exposure concentration
1.5.2.3. Negative and positive controls
1.5.3. Procedure
1.5.4. Incubation
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
TEST REPORT
4. REFERENCES
B.15. MUTAGENICITY TESTING AND SCREENING FOR CARCINOGENICITY GENE MUTATION —
SACCHAROMYCES CEREVISIAE
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
Preparations
Metabolic activation
Test conditions
Tester strains
Media
Use of negative and positive controls
Exposure concentration
Incubation conditions
Spontaneous mutation frequencies
Number of replicates
Procedure
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.16. MITOTIC RECOMBINATION —
SACCHAROMYCES CEREVISIAE
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
Preparations
Metabolic activation
Test conditions
Tester strains
Media
Use of negative and positive controls
Exposure concentrations
Incubation conditions
Spontaneous mitotic recombination frequencies
Number of replicates
Procedures
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.17. MUTAGENICITY —
IN VITRO
MAMMALIAN CELL GENE MUTATION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations
1.4.1.1. Cells
1.4.1.2. Media and culture conditions
1.4.1.3. Preparation of cultures
1.4.1.4. Metabolic activation
1.4.1.5. Test substance/Preparation
1.4.2. Test conditions
1.4.2.1. Solvent/Vehicle
1.4.2.2. Exposure concentrations
1.4.2.3. Controls
1.4.3. Procedure
1.4.3.1. Treatment with the test substance
1.4.3.2. Measurement of survival, viability and mutant frequency
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
TEST REPORT
4. REFERENCES
B.18. DNA DAMAGE AND REPAIR — UNSCHEDULED DNA SYNTHESIS — MAMMALIAN CELLS
IN VITRO
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
Preparations
Test conditions
Number of cultures
Use of negative and positive controls
Exposure concentrations
Cells
Metabolic activation
Procedure
Preparation of cultures
Treatment of the cultures with the test substance
Primary rat hepatocytes
Established cell lines and lymphocytes
Analysis
Autoradiographic determinations
LSC determinations
2. DATA
2.1. AUTORADIOGRAPHIC DETERMINATIONS
2.2. LSC DETERMINATIONS
3. REPORTING
3.1. TEST REPORT
3.2. VALUATION AND INTERPRETATION
4. REFERENCES
B.19. SISTER CHROMATID EXCHANGE ASSAY
IN VITRO
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Preparations
1.6.2.
Test conditions
Number of cultures
Use of negative and positive controls
Exposure concentrations
1.6.3.
Procedure
Preparation of cultures
Treatment
Harvesting of cells
Chromosome preparation and staining
Analysis
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.20. SEX-LINKED RECESSIVE LETHAL TEST IN
DROSOPHILA MELANOGASTER
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLES OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
Preparations
Stocks
Test substance
Number of animals
Route of administration
Use of negative and positive controls
Exposure levels
Procedure
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.21.
IN VITRO
MAMMALIAN CELL TRANSFORMATION TESTS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
Preparations
Cells
Medium
Test substance
Metabolic activation
Test conditions
Use of negative and positive controls
Exposure concentrations
Procedure
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.22. RODENT DOMINANT LETHAL TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
Preparations
Test conditions
Route of administration
Experimental animals
Number and sex
Use of negative and positive controls
Dose levels
Limit test
Procedure
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.23. MAMMALIAN SPERMATOGONIAL CHROMOSOME ABERRATION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations
1.4.1.1. Selection of animal species
1.4.1.2. Housing and feeding conditions
1.4.1.3. Preparation of the animals
1.4.1.4. Preparation of doses
1.4.2. Test conditions
1.4.2.1. Solvent/Vehicle
1.4.2.2. Controls
1.5. PROCEDURE
1.5.1. Number of animals
1.5.2. Treatment schedule
1.5.3. Dose levels
1.5.4. Limit test
1.5.5. Administration of doses
1.5.6. Chromosome preparation
1.5.7. Analysis
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
TEST REPORT
4. REFERENCES
B.24. MOUSE SPOT TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
Preparations
Experimental animals
Number and sex
Use of negative and positive controls
Route of administration
Dose levels
Procedure
Analysis
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.25. MOUSE HERITABLE TRANSLOCATION
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
Preparations
Route of administration
Experimental animals
Number of animals
Use of negative and positive controls
Dose levels
Procedure
Treatment and mating
Testing for translocation heterozygosity
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.26. SUB-CHRONIC ORAL TOXICITY TESTREPEATED DOSE 90 – DAY ORAL TOXICITY STUDY IN RODENTS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations of animals
1.4.2. Preparations of doses
1.4.3. Test conditions
1.4.3.1. Experimental animals
1.4.3.2. Number and sex
1.4.3.3. Dose levels
1.4.3.4. Limit test
1.5. PROCEDURE
1.5.1. Administration of doses
1.5.2. Observations
1.5.2.1. Body weight and food/water consumption
1.5.2.2. Haematology and clinical biochemistry
1.5.2.3. Gross necropsy
1.5.2.4. Histopathology
2. DATA AND REPORTING
2.1. DATA
2.2. TEST REPORT
2.2.1. Test substance:
2.2.2. Test species:
2.2.3. Test conditions:
2.2.4. Results:
3. REFERENCES
B.27. SUB-CHRONIC ORAL TOXICITY TEST REPEATED DOSE 90-DAY ORAL TOXICITY STUDY IN NON-RODENTS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Selection of animal species
1.4.2. Preparation of animals
1.4.3. Preparations of doses
1.5. PROCEDURE
1.5.1. Number and sex of animals
1.5.2. Dosage
1.5.3. Limit test
1.5.4. Administration of doses
1.5.5. Observations
1.5.5.1. Body weight and food/water consumption
1.5.5.2. Haematology and clinical biochemistry
1.5.5.3. Gross necropsy
1.5.5.4. Histopathology
2. DATA AND REPORTING
2.1. DATA
2.2. TEST REPORT
2.2.1. Test substance:
2.2.2. Test species:
2.2.3. Test conditions:
2.2.4. Results:
B.28. SUB-CHRONIC DERMAL TOXICITY STUDY 90-DAY REPEATED DERMAL DOSE STUDY USING RODENT SPECIES
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Preparations
1.6.2.
Test conditions
1.6.2.1.
Experimental animals
1.6.2.2.
Number and sex
1.6.2.3.
Dose levels
1.6.3.
Limit test
1.6.4.
Observation period
1.6.5.
Procedure
Gross necropsy
Histopathological examination
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.29. SUB-CHRONIC INHALATION TOXICITY STUDY 90-DAY REPEATED INHALATION DOSE STUDY USING RODENT SPECIES
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Preparations
1.6.2.
Test conditions
1.6.2.1.
Experimental animals
1.6.2.2.
Number and sex
1.6.2.3.
Exposure concentrations
1.6.2.4.
Exposure time
1.6.2.5.
Equipment
1.6.2.6.
Observation period
1.6.3.
Procedure
Gross necropsy
Histopathological examination
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.30. CHRONIC TOXICITY TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Preparations
1.6.2.
Test conditions
1.6.2.1.
Experimental animals
1.6.2.2.
Number and sex
1.6.2.3.
Dose levels and frequency of exposure
1.6.2.4.
Controls
1.6.2.5.
Route of administration
1.6.2.6.
Oral studies
1.6.2.7.
Inhalation studies
1.6.2.8.
Exposure chambers
1.6.2.9.
Duration of study
1.6.3.
Procedure
Observations
Haematological examination
Urinalysis
Clinical chemistry
Gross necropsy
Histopathology
2. DATA
3. REPORTING
TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.31. PRENATAL DEVELOPMENTAL TOXICITY STUDY
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCE
1.4. PRINCIPLE OF THE TEST METHOD
1.5. DESCRIPTION OF THE TEST METHOD
1.5.1. Selection of animal species
1.5.2. Housing and feeding conditions
1.5.3. Preparation of the animals
1.6. PROCEDURE
1.6.1. Number and sex of animals
1.6.2. Preparation of doses
1.6.3. Dosage
1.6.4. Limit test
1.6.5. Administration of doses
1.6.6. Observations of the dams
1.6.7. Body weight and food consumption
1.6.8. Post-mortem examination
1.6.9. Examination of uterine contents
1.6.10. Examination of foetuses
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION OF RESULTS
2.3. INTERPRETATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
B.32. CARCINOGENICITY TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Experimental animals
1.6.2.
Number and sex
1.6.3.
Dose levels and frequency of exposure
1.6.4.
Controls
1.6.5.
Route of administration
1.6.5.1. Oral studies
1.6.5.2. Dermal studies
1.6.5.3. Inhalation studies
1.6.6.
Exposure chambers
1.6.7.
Duration of study
1.6.8.
Procedure
1.6.8.1.
Observations
1.6.8.2.
Clinical examinations
Haematology
Gross necropsy
Histopathology
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.33. COMBINED CHRONIC TOXICITY/CARCINOGENICITY TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Experimental animals
1.6.2.
Number and sex
1.6.3.
Dose levels and frequency of exposure
1.6.4.
Controls
1.6.5.
Route of administration
1.6.5.1. Oral tests
1.6.5.2. Dermal tests
1.6.5.3. Inhalation tests
1.6.6.
Exposure chambers
1.6.7.
Duration of test
1.6.8.
Procedure
1.6.8.1.
Observations
1.6.8.2.
Clinical examinations
Haematology
Urinalysis
Clinical chemistry
Gross necropsy
Histopathology
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.34. ONE-GENERATION REPRODUCTION TOXICITY TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Preparations
1.6.2.
Experimental animals
Selection of species
Number and sex
1.6.3.
Test conditions
1.6.3.1.
Dose levels
1.6.3.2.
Limit test
1.6.3.3.
Performance of the test
Experimental schedules
Mating procedure
Litter sizes
1.6.4.
Observations
1.6.5.
Pathology
1.6.5.1.
Necropsy
1.6.5.2.
Histopathology
2. DATA
3. REPORTING
TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.35. TWO-GENERATION REPRODUCTION TOXICITY STUDY
1. METHOD
1.1. INTRODUCTION
1.2. PRINCIPLE OF THE TEST METHOD
1.3. DESCRIPTION OF THE TEST METHOD
1.3.1. Selection of animal species
1.3.2. Housing and feeding conditions
1.3.3. Preparation of animals
1.4. PROCEDURE
1.4.1. Number and sex of animals
1.4.2. Preparation of doses
1.4.3. Dosage
1.4.4. Limit test
1.4.5. Administration of doses
1.4.6. Experimental schedules
1.4.7. Mating procedure
1.4.7.1. Parental (P) mating
1.4.7.2. F1 mating
1.4.7.3. Second mating
1.4.7.4. Litter size
1.5. OBSERVATIONS
1.5.1. Clinical observations
1.5.2. Body weight and food/water consumption of parent animals
1.5.3. Oestrus cycle
1.5.4. Sperm parameters
1.5.5. Offspring
1.5.6. Gross necropsy
1.5.7. Organ weights
1.5.8. Histopathology
1.5.8.1. Parental animals
1.5.8.2. Weanlings
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION OF RESULTS
2.3. INTERPRETATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
B.36. TOXICOKINETICS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Preparations
1.6.2. Test conditions
1.6.2.1. Experimental animals
1.6.2.2. Number and sex
1.6.3. Dose levels
1.6.4. Route of administration
1.6.5. Observation period
1.6.6. Procedure
Absorption
Distribution
Excretion
Metabolism
2. DATA
3. REPORTING
3.1. TEST REPORT
3.2. EVALUATION AND INTERPRETATION
4. REFERENCES
B.37. DELAYED NEUROTOXICITY OF ORGANOPHOSPHORUS SUBSTANCES FOLLOWING ACUTE EXPOSURE
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. DESCRIPTION OF THE TEST METHOD
1.5.1. Preparations
1.5.2. Test conditions
1.5.2.1. Test animals
1.5.2.2. Number and sex
1.5.2.3. Dose levels
1.5.2.4. Limit test
1.5.3. Observation period
1.5.4. Procedure
General observation
Body weight
Biochemistry
Gross necropsy
Histopathological examination
2. DATA
3. REPORTING
TEST REPORT
4. REFERENCES
B.38. DELAYED NEUROTOXICITY OF ORGANOPHOSPHORUS SUBSTANCES 28-DAY REPEATED DOSE STUDY
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations
1.4.2. Test conditions
1.4.2.1. Test animals
1.4.2.2. Number and sex
1.4.2.3. Dose levels
1.4.2.4. Limit test
1.4.2.5. Observation period
1.4.3. Procedure
General observations
Body weight
Biochemistry
Gross necropsy
Histopathological examination
2. DATA
3. REPORTING
TEST REPORT
4. REFERENCES
B.39. UNSCHEDULED DNA SYNTHESIS (UDS) TEST WITH MAMMALIAN LIVER CELLS IN VIVO
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE METHOD
1.4.1. Preparations
1.4.1.1. Selection of animal species
1.4.1.2. Housing and feeding conditions
1.4.1.3. Preparation of the animals
1.4.1.4. Test substance/Preparation
1.4.2. Test conditions
1.4.2.1. Solvent/Vehicle
1.4.2.2. Controls
1.5. PROCEDURE
1.5.1. Number and sex of animals
1.5.2. Treatment schedule
1.5.3. Dose levels
1.5.4. Limit test
1.5.5. Administration of doses
1.5.6. Preparation of liver cells
1.5.7. Determination of UDS
1.5.8. Analysis
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
TEST REPORT
4. REFERENCES
B.40.
IN VITRO
SKIN CORROSION: TRANSCUTANEOUS ELECTRICAL RESISTANCE TEST (TER)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. DESCRIPTION OF THE TEST METHOD
1.5.1. Animals
1.5.2. Preparation of the skin discs
1.5.3. Application of the test and control substances
1.5.4. TER measurements
1.5.5. Dye binding methods
1.5.5.1. Sulforhodamine B dye application and removal
1.5.5.2. Calculation of dye content
2. DATA
2.1. INTERPRETATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Figure 1
Apparatus for the rat skin TER assay
Figure 2
Dimensions of the polytetrafluoroethylene (PFTE) and receptor tubes and electrodes used
B.40 BIS.
IN VITRO
SKIN CORROSION: HUMAN SKIN MODEL TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.4.1. Procedure
1.4.1.1. Human skin models
1.4.1.1.1.
General model conditions:
1.4.1.1.2.
Functional model conditions:
1.4.1.2. Application of the test and control substances
1.4.1.3. Cell viability measurements
2. DATA
2.1. INTERPRETATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
B.41.
IN VITRO
3T3 NRU PHOTOTOXICITY TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Preparations
1.4.1.1. Cells
1.4.1.2. Media and culture conditions
1.4.1.3. Preparation of cultures
1.4.1.4. Preparation of test substance
1.4.1.5. Irradiation conditions
1.4.1.5.1.
Light source
1.4.1.5.2.
Dosimetry
1.4.2. Test conditions
1.4.2.1. Test substance concentrations
1.4.2.2. Controls
1.4.2.2.1.
Radiation sensitivity of the cells, establishing of historical data:
1.4.2.2.2.
Radiation sensitivity, check of current test:
1.4.2.2.3.
Viability of solvent controls:
1.4.2.2.4.
Positive control:
1.4.3. Test procedure (6)(7)(8)(16)(17):
1.4.3.1. 1st day:
1.4.3.2. 2nd day:
1.4.3.3. 3rd day:
1.4.3.3.1.
Microscopic evaluation
1.4.3.3.2.
Neutral Red uptake test
2. DATA
2.1. QUALITY AND QUANTITY OF DATA
2.2. EVALUATION OF RESULTS
2.3. INTERPRETATION OF RESULTS
2.4. INTERPRETATION OF DATA
3. REPORTING
TEST REPORT
4. REFERENCES
Appendix 1
Role of the 3T3 NRU PT in a sequential approach to the phototoxicity testing of chemicals
Appendix 2
Figure 1
Spectral power distribution of a filtered solar simulator
Figure 2
Irradiation sensivity of Balb/c 3T3 cells (as measured in the UVA range)
B.42. SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY
1. METHOD
1.1. INTRODUCTION
1.2. PRINCIPLE OF THE TEST METHOD
1.3. DESCRIPTION OF THE TEST METHOD
1.3.1. Preparations
1.3.1.1. Housing and feeding conditions
1.3.1.2. Preparation of animals
1.3.2. Test conditions
1.3.2.1. Experimental animals
1.3.2.2. Reliability check
1.3.2.3. Number of animals, dose levels and vehicle selection.
1.3.3. Test procedure
1.3.3.1. Experimental schedule
1.3.3.2. Preparation of cell suspensions
1.3.3.3. Determination of cell proliferation (incorporated radioactivity)
1.3.3.4. Observations
1.3.3.4.1.
Clinical observations
1.3.3.4.2.
Body weights
1.3.4. Calculation of results
2. DATA
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
B.43. NEUROTOXICITY STUDY IN RODENTS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Selection of animal species
1.4.2. Housing and feeding conditions
1.4.3. Preparation of animals
1.4.4. Route of administration and preparation of doses
1.5. PROCEDURES
1.5.1. Number and sex animals
1.5.2. Treatment and control group
1.5.3. Reliability check
1.5.4. Dose selection
1.5.5. Limit test
1.5.6. Administration of doses
1.6. OBSERVATION
1.6.1. Frequency of observations and tests
1.6.1.1. Observations of general health condition and mortality/morbidity
1.6.1.2. Detailed clinical observations
1.6.1.3. Functional tests
1.6.2. Body weight and food/water consumption
1.6.3. Ophthalmology
1.6.4. Haematology and clinical biochemistry
1.6.5. Histopathology
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
B.44. SKIN ABSORPTION:
IN VIVO
METHOD
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE METHOD
1.4.1. Selection of animal species
1.4.2. Number and sex of animals
1.4.3. Housing and feeding conditions
1.4.4. Preparation of animals
1.4.5. Test substance
1.4.6. Test preparation
1.4.7. Application to the skin
1.4.8. Duration of exposure and sampling
1.4.9. Terminal procedures
1.4.10. Analysis
2. DATA
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Figure 1
An example of a design of a typical device used to define and protect dermal application site during
in vivo
percutaneous absorption studies
B.45. SKIN ABSORPTION:
IN VITRO
METHOD
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. DESCRIPTION OF THE TEST METHOD
1.4.1. Diffusion cell
1.4.2. Receptor fluid
1.4.3. Skin preparations
1.4.4. Skin preparation integrity
1.4.5. Test substance
1.4.6. Test preparation
1.4.7. Test substances concentrations and formulations
1.4.8. Application to the skin
1.4.9. Temperature
1.4.10. Duration of exposure and sampling
1.4.11. Terminal procedures
1.4.12. Analysis
2. DATA
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Figure 1
An example of a typical design of a static diffusion cell for
in vitro
percutaneous absorption studies
PART C: METHODS FOR THE DETERMINATION OF ECOTOXICITY
C.1. ACUTE TOXICITY FOR FISH
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Reagents
1.6.1.1. Solutions of test substances
1.6.1.2. Holding and dilution water
1.6.2. Apparatus
1.6.3. Test fish
1.6.3.1. Holding
1.6.3.2. Mortality
1.6.4. Adaptation
1.6.5. Test procedure
Limit test
2. DATA AND EVALUATION
3. REPORTING
4. REFERENCES
Appendix 1
Reconstituted water
Example of a suitable dilution water
Stock solutions
Reconstituted dilution water
Appendix 2
Fish species recommended for testing
Collection
Appendix 3
Example of concentration: percentage mortality
C.2.
DAPHNIA
SP. ACUTE IMMOBILISATION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. INFORMATION ON THE TEST SUBSTANCE
1.5. REFERENCE SUBSTANCES
1.6. QUALITY CRITERIA
1.7. DESCRIPTION OF THE TEST METHOD
1.7.1. Apparatus
1.7.2. Test organism
1.7.3. Holding and dilution water
1.7.4. Test solutions
1.8. PROCEDURE
1.8.1. Conditions of exposure
1.8.1.1. Test groups and controls
1.8.1.2. Test concentrations
1.8.1.3. Incubation conditions
1.8.1.4. Duration
1.8.2. Observations
1.8.3. Analytical measurements
1.9. LIMIT TEST
2. DATA
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Appendix 1
SOME CHEMICAL CHARACTERISTICS OF AN ACCEPTABLE DILUTION WATER
Appendix 2
EXAMPLES OF SUITABLE RECONSTITUTED TEST WATER
ISO Test water (1)
Elendt M7 and M4 medium
Acclimation to Elendt M4 and M7 medium
Preparation
Trace element
M4 and M7 media
C.3. ALGAL INHIBITION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST PROCEDURE
1.6.1. Reagents
1.6.1.1. Solutions of test substances
1.6.1.2. Test medium
1.6.2. Apparatus
1.6.3. Test organisms
1.6.4. Test procedure
Initial cell density
Concentrations of test substance
Replicates and controls
Performance of the test
Testing volatile substances
Limit test
2. DATA AND EVALUATION
2.1. COMPARISON OF AREAS UNDER THE GROWTH CURVES
2.2. COMPARISON OF GROWTH RATES
2.3. CALCULATION OF THE NOEC
3. REPORTING
4. REFERENCES
Appendix 1
Example of a procedure for the culturing of algae
General observations
Procedures for obtaining algal cultures
Preparation of nutrient solutions (media):
Stock culture:
Pre-culture:
Appendix 2
C.4. DETERMINATION OF ‘READY’ BIODEGRADABILITY
PART I. GENERAL CONSIDERATIONS
I.1. INTRODUCTION
I.2. SELECTION OF THE APPROPRIATE METHOD
I.3. REFERENCE SUBSTANCES
I.4. PRINCIPLE OF THE TEST METHODS
I.5. QUALITY CRITERIA
I.5.1. Reproducibility
I.5.2. Validity of the test
I.6. GENERAL PROCEDURES AND PREPARATIONS
I.6.1. Dilution water
I.6.2. Stock solutions of mineral components
Stock solutions:
I.6.3. Stock solutions of chemicals
I.6.4. Inocula
I.6.4.1. Inoculum from activated sludges
I.6.4.2. Other sources of inoculum
I.6.5. Pre-conditioning of inocula
I.6.6. Abiotic controls
I.6.7. Number of flasks
I.7. DATA AND EVALUATION
I.7.1. Degradation measured by means of DOC determination
I.7.2. Degradation measured by means of specific analysis
1.7.3. Abiotic degradation
I.8. REPORTING
PART II. DOC DIE-AWAY TEST
(Method C.4-A)
II.1. PRINCIPLE OF THE METHOD
II.2. DESCRIPTION OF THE METHOD
II.2.1. Apparatus
II.2.2. Preparation of mineral medium
II.2.3. Preparation and pre-conditioning of inoculum
II.2.4. Preparation of flasks
II.2.5. Number of flasks in typical run
II.2.6. Performance of the test
II.3. DATA AND REPORTING
II.3.1. Treatment of results
II.3.2. Validity of results
II.3.3. Reporting
II.4. DATA SHEET
DOC DIE-AWAY TEST
3. TEST SUBSTANCE
4. INOCULUM
5. CARBON DETERMINATIONS
6. EVALUATION OF RAW DATA
7. ABIOTIC CONTROL
(optional)
8. SPECIFIC CHEMICAL ANALYSIS
(optional)
PART III. MODIFIED OECD SCREENING TEST
(Method C.4-B)
III.1. PRINCIPLE OF THE METHOD
III.2. DESCRIPTION OF THE METHOD
III.2.1.
Apparatus
III.2.2. Preparation of mineral medium
III.2.3. Preparation and pre-conditioning of inoculum
III.2.4. Preparation of flasks
III.2.5. Number of flasks in typical run
III.2.6. Performance of the test
III.3. DATA AND REPORTING
III.3.1. Treatment of results
III.3.2.
Validity of results
III.3.3.
Reporting
III.4. DATA SHEET
MODIFIED OECD SCREENING TEST
3. TEST SUBSTANCE
4. INOCULUM
5.
CARBON DETERMINATIONS
6. EVALUATION OF RAW DATA
7. ABIOTIC CONTROL
(optional)
8. SPECIFIC CHEMICAL ANALYSIS
(optional)
PART IV.
CO
2
EVOLUTION TEST (Method C.4-C)
IV.1. PRINCIPLE OF THE METHOD
IV.2. DESCRIPTION OF THE METHOD
IV.2.1. Apparatus
IV.2.2. Preparation of mineral medium
IV.2.3. Preparation and pre-conditioning of inoculum
IV.2.4. Preparation of flasks
IV.2.5. Number of flasks in a typical run
IV.2.6. Performance of the test
IV.3. DATA AND REPORTING
IV.3.1. Treatment of results
IV.3.2. Validity of results
IV.3.3. Reporting
IV.4. DATA SHEET
CARBON DIOXIDE EVOLUTION TEST
3. TEST SUBSTANCE
4. INOCULUM
6. CARBON ANALYSIS
(optional)
7. ABIOTIC DEGRADATION
(optional)
PART V. MANOMETRIC RESPIROMETRY TEST
(Method C.4-D)
V.1. PRINCIPLE OF THE METHOD
V.2. DESCRIPTION OF THE METHOD
V.2.1. Apparatus
V.2.2. Preparation of mineral medium
V.2.3. Preparation and pre-conditioning of inoculum
V.2.4. Preparation of flasks
V.2.5. Number of flasks in a typical run
V.2.6. Performance of the test
V.3. DATA AND REPORTING
V.3.1. Treatment of results
V.3.2. Validity of results
V.3.3. Reporting
V.4.
DATA SHEET
MANOMETRIC RESPIROMETRY TEST
3. TEST SUBSTANCE
4. INOCULUM
5. OXYGEN UPTAKE: BIODEGRADABILITY
6. CORRECTION FOR NITRIFICATION
(see Annex V)
7. CARBON ANALYSIS
(optional)
8. SPECIFIC CHEMICAL
(optional)
9. ABIOTIC DEGRADATION
(optional)
PART VI. CLOSED BOTTLE TEST
(Method C.4-E)
VI.1 PRINCIPLE OF THE TEST METHOD
VI.2 DESCRIPTION OF THE METHOD
VI.2.1. Apparatus
VI.2.2. Preparation of mineral medium
VI.2.3. Preparation of the inoculum
VI.2.4. Preparation of flasks
VI.2.5. Number of flasks in a typical run
VI.2.6.
Performance of the test
VI.3. DATA AND REPORTING
VI.3.1. Treatment of results
VI.3.2. Validity of results
VI.3.3. Reporting
VI.4. DATA SHEET
CLOSED BOTTLE TEST
3. TEST SUBSTANCE
4. INOCULUM
5. DO DETERMINATION
6. CORRECTION FOR NITRIFICATION
(see Annex V)
7. DO DEPLETION: % DEGRADATION
8. BLANK DO DEPLETIONS
PART VII. M.I.T.I. TEST
(Method C.4-F)
VII.1. PRINCIPLE OF THE METHOD
VII.2. DESCRIPTION OF THE METHOD
VII.2.1. Apparatus
VII.2.2. Preparation of mineral medium
VII.2.3. Preparation of inoculum
VII.2.4. Preparation of flasks
VII.2.5. Performance of the test
VII.3. DATA AND REPORTING
VII.3.1. Treatment of results
VII.3.2. Validity of results
VII.3.3. Reporting
VII.4. DATA SHEET
MITI (I) TEST
3. TEST SUBSTANCE
4. INOCULUM
5. OXYGEN UPTAKE: BIODEGRADABILITY
6. CARBON ANALYSIS
(optional)
7. SPECIFIC CHEMICAL ANALYTICAL DATA
8. REMARKS
Appendix 1
ABBREVIATIONS AND DEFINITIONS
Primary biodegradation:
Ultimate biodegradation (aerobic):
Readily biodegradable:
Inherently biodegradable:
Treatability:
Lag time
Degradation time
10-day window
Appendix 2
CALCULATION AND DETERMINATION OF SUITABLE SUMMARY PARAMETERS
1. Carbon content
2. Theoretical oxygen demand (ThOD)
3. Chemical Oxygen Demand (COD)
4. Dissolved organic carbon (DOC)
Remarks:
BIBLIOGRAPHY
Appendix 3
EVALUATION OF THE BIODEGRADABILITY OF POORLY SOLUBLE SUBSTANCES
BIBLIOGRAPHY
Appendix 4
EVALUATION OF THE BIODEGRADABILITY OF CHEMICALS SUSPECTED TO BE TOXIC TO THE INOCULUM
BIBLIOGRAPHY
Appendix 5
CORRECTION FOR OXYGEN UPTAKE FOR INTERFERENCE BY NITRIFICATION
C.5 DEGRADATION — BIOCHEMICAL OXYGEN DEMAND
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
2. DATA AND EVALUATION
3. REPORTING
4.
REFERENCES
C.6. DEGRADATION — CHEMICAL OXYGEN DEMAND
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
2. DATA AND EVALUATION
3. REPORTING
4. REFERENCES
C.7. DEGRADATION — ABIOTIC DEGRADATION: HYDROLYSIS AS A FUNCTION OF PH
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. APPLICABILITY OF THE METHOD
1.4. PRINCIPLE OF THE TEST METHOD
1.5. INFORMATION ON THE TEST SUBSTANCE
1.6. REFERENCE SUBSTANCES
1.7. QUALITY CRITERIA
1.7.1. Recovery
1.7.2. Repeatability and sensitivity of analytical method
1.7.3. Confidence intervals for hydrolysis kinetic data
1.8. DESCRIPTION OF THE TEST METHOD
1.8.1. Equipment and apparatus
1.8.2. Test substance application
1.8.3. Buffer solutions
1.8.4. Test conditions
1.8.4.1. Test temperature
1.8.4.2. Light and oxygen
1.8.4.3. Test duration
1.8.5. Performance of the test
1.8.5.1. Preliminary test (Tier 1)
1.8.5.2. Hydrolysis of unstable substances (Tier 2)
1.8.5.3. Identification of hydrolysis products (Tier 3)
1.8.5.4. Optional tests
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Appendix 1
Tiered hydrolysis test scheme
Appendix 2
Definitions and units
Appendix 3
Buffer Systems
A. CLARK AND LUBS:
Buffer mixtures of CLARK and LUBS
(14)
Buffer mixtures of CLARK and LUBS (Continued)
B. KOLTHOFF AND VLEESCHHOUWER:
Citrate buffers of KOLTHOFF and VLEESCHHOUWER
C. SÖRENSEN:
Borate mixtures of SÖRENSEN
Phosphate mixtures of SÖRENSEN
C.8. TOXICITY FOR EARTHWORMS
ARTIFICIAL SOIL TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITION AND UNIT
1.3. REFERENCE SUBSTANCE
1.4. PRINCIPLE OF THE TEST
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Materials
1.6.1.1.
Test substrate
1.6.1.2.
Test containers
1.6.2.
Test conditions
1.6.3.
Test procedure
Test concentrations
Range finding test
Definitive test
Mixture of the basic test substrate and the test substance
1.6.4.
Test organisms
2. DATA
2.1. TREATMENT AND EVALUATION OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Appendix
Breeding and keeping of the worms before testing
Keeping and breeding conditions
C.9. BIODEGRADATION
ZAHN-WELLENS TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST PROCEDURE
1.6.1.
Preparations
1.6.1.1.
Reagents
1.6.1.2.
Apparatus
1.6.1.3.
Preparation of the inoculum
1.6.1.4.
Preparation of the test solutions
1.6.2.
Performance of the test
Functional control of activated sludge
Adaptation
Analytical means
2. DATA AND EVALUATION
3. REPORTING
3.1. TEST REPORT
3.2. INTERPRETATION OF RESULTS
4. REFERENCES
Appendix
EVALUATION EXAMPLE
Figure 1
Examples of biodegradation curves
Figure 2
Examples of sludge adaptation
C.10. BIODEGRADATION
ACTIVATED SLUDGE SIMULATION TESTS
1. METHOD
1.1. INTRODUCTION
1.1.1.
General remarks
1.1.2.
Determination of ultimate biodegradability (DOC/COD analysis)
1.1.3.
Determination of primary biodegradability (specific analysis)
1.2. DEFINITIONS AND UNITS
1.2.1.
DOC/COD analysis
1.2.2.
Specific analysis
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHODS
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Preparation
1.6.1.1.
Apparatus
1.6.1.2.
Filtration
1.6.1.3.
Sewage
1.6.1.4.
Stock solution of test material
1.6.1.5.
Inoculum
1.6.2.
Procedure
1.6.2.1.
Running-in period: Sludge formation/stabilisation of the units
1.6.2.2.
Test procedure
1.6.2.3.
Analysis
2. DATA AND EVALUATION
2.1. COUPLED UNITS MODE
2.2. NON-COUPLED UNITS MODE
2.2.1.
Using COD/DOC determinations
2.2.2.
Using specific analysis
3. REPORTING
3.1. TEST REPORT
3.2. INTERPRETATION OF RESULTS
4. REFERENCES
Appendix 1
Figure 1
Figure 2
Appendix 2
Figure 1
Equipment used for assessing biodegradability
Figure 2
Details of three-litre porous-pot aeration vessel
Appendix 3
Operating conditions for the Activated Sludge Simulation Test
Check in each group
Apparatus
Mode of operation
Transinoculation
Mean retention time
Base nutrient
Inoculum
Test material addition
Analysis
C.11. BIODEGRADATION
ACTIVATED SLUDGE RESPIRATION INHIBITION TEST
1. METHOD
1.1. INTRODUCTION
Recommendation
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1.
Reagents
1.6.1.1.
Solutions of the test substance
1.6.1.2.
Solution of control substance
1.6.1.3.
Synthetic sewage
1.6.2.
Apparatus
1.6.3.
Preparation of the inoculum
1.6.4.
Performance of the test
2. DATA AND EVALUATION
3. REPORTING
3.1. TEST REPORT
3.2. INTERPRETATION OF DATA
4. REFERENCES
C.12. BIODEGRADATION
MODIFIED SCAS TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. QUALITY CRITERIA
1.6. DESCRIPTION OF THE TEST PROCEDURE
1.6.1.
Preparations
1.6.2.
Test conditions
1.6.3.
Performance of the test
2. DATA AND EVALUATION
3. REPORTING
3.1. TEST REPORT
3.2. INTERPRETATION OF RESULTS
4. REFERENCES
Appendix 1
SCAS test: example of results
Appendix 2
Example of test apparatus
Figure 1
C.13 BIOCONCENTRATION: FLOW-THROUGH FISH TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. INFORMATION ON THE TEST SUBSTANCE
1.5. VALIDITY OF THE TEST
1.6. REFERENCE COMPOUNDS
1.7. DESCRIPTION OF THE TEST METHOD
1.7.1.
Apparatus
1.7.2.
Water
1.7.3.
Test Solutions
1.7.4.
Selection of species
1.7.5.
Holding of fish
1.8. PERFORMANCE OF THE TEST
1.8.1.
Preliminary test
1.8.2.
Conditions of exposure
1.8.2.1.
Duration of uptake phase
1.8.2.2.
Duration of the depuration phase
1.8.2.3.
Numbers of test fish
1.8.2.4.
Loading
1.8.2.5.
Feeding
1.8.2.6.
Light and temperature
1.8.2.7.
Test concentrations
1.8.2.8.
Controls
1.8.3.
Frequency of water quality measurements
1.8.4.
Sampling and analysis of fish and water
1.8.4.1.
Fish and water sampling schedule
1.8.4.2.
Sampling and Sample Preparation
1.8.4.3.
Quality of Analytical method
1.8.4.4.
Analysis of Fish Sample
2. DATA
2.1. TREATMENT OF RESULTS
2.2. INTERPRETATION OF RESULTS
3. REPORTING
3.1. TEST SUBSTANCE:
3.2. TEST SPECIES
3.3. TEST CONDITIONS:
3.4. RESULTS:
4. REFERENCES
Appendix 1
Chemical characteristics of an acceptable dilution water
Appendix 2
Fish species recommended for testing
Collection
Appendix 3
Prediction of the duration of the uptake and depuration phases
1. Prediction of the duration of the uptake phase
2. Prediction of the duration of the depuration phase
Literature
(of Appendix 3)
Appendix 4
Theoretical example of sampling schedule for bioconcentration tests of substances with log P
ow
= 4
Appendix 5
Model discrimination
Graphical method for determination of depuration (loss) rate constant k
2
Graphical method for determination of uptake rate constant k
1
Computer method for calculation of uptake and depuration (loss) rate constants
C.14. FISH JUVENILE GROWTH TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. INFORMATION ON THE TEST SUBSTANCE
1.5. VALIDITY OF THE TEST
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Apparatus
1.6.2. Water
1.6.3. Test solutions
1.6.4. Selection of species
1.6.5. Holding of fish
1.7. TEST DESIGN
1.7.1. Design for analysis by regression
1.7.2. Design for estimation of a NOEC/LOEC using Analysis of Variance (ANOVA)
1.8. PROCEDURE
1.8.1. Selection and weighing of test fish
1.8.2. Conditions of exposure
1.8.2.1. Duration
1.8.2.2. Loading rates and stocking densities
1.8.2.3. Feeding
1.8.2.4. Light and temperature
1.8.3. Test concentrations
1.8.4. Controls
1.8.5. Frequency of analytical determinations and measurements
1.8.6. Observations
2. DATA AND REPORTING
2.1. TREATMENT OF RESULTS
2.1.1. Analysis of results by regression (concentration-response modelling)
2.1.2. Analysis of results for the estimation of the LOEC
2.2. INTERPRETATION OF RESULTS
2.3. TEST REPORT
2.3.1. Test substance:
2.3.2. Test species:
2.3.3. Test conditions:
2.3.4. Results:
3.
REFERENCES
Appendix 1
FISH SPECIES RECOMMENDED FOR TESTING AND SUITABLE TEST CONDITIONS
Appendix 2
SOME CHEMICAL CHARACTERISTICS OF AN ACCEPTABLE DILUTION WATER
Appendix 3
Logarithmic series of concentrations suitable for toxicity test (9)
C.15. FISH, SHORT-TERM TOXICITY TEST ON
EMBRYO
AND SAC-FRY STAGES
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST
1.4. INFORMATION ON THE TEST SUBSTANCE
1.5. VALIDITY OF THE TEST
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Test chambers
1.6.2. Selection of fish species
1.6.3. Holding of the brood fish
1.6.4. Handling of embryos and larvae
1.6.5. Water
1.6.6. Test solutions
1.7. PROCEDURE
1.7.1. Conditions of exposure
1.7.1.1. Duration
1.7.1.2. Loading
1.7.1.3. Light and temperature
1.7.2. Test concentrations
1.7.3. Controls
1.7.4. Frequency of analytical determinations and measurements
1.7.5. Observations
1.7.5.1. Stage of embryonic development
1.7.5.2. Hatching and survival
1.7.5.3. Abnormal appearance
1.7.5.4. Abnormal behaviour
1.7.5.5. Length
1.7.5.6. Weight
2. DATA AND REPORTING
2.1. TREATMENT OF RESULTS
2.2. INTERPRETATION OF RESULTS
2.3. THE TEST REPORT
2.3.1. Test substance:
2.3.2. Test species:
2.3.3. Test conditions:
2.3.4. Results:
3. REFERENCES
Appendix 1
GUIDANCE ON PERFORMANCE OF A TOXICITY TEST ON EMBRYOS AND SAC-FRY OF ZEBRAFISH
(BRACHYDANIO RERIO)
INTRODUCTION
CONDITIONS OF PARENTAL FISH, REPRODUCTION AND EARLY-LIFE STAGES
CALCULATIONS AND STATISTICS
LC
50
AND EC
50
DETERMINATIONS
ESTIMATION OF LOEC AND NOEC
REFERENCES
Appendix 2
TEST CONDITIONS, DURATION AND SURVIVAL CRITERIA FOR RECOMMENDED SPECIES
Appendix 3
Test conditions, duration and survival criteria for other well documented species
Appendix 4
SOME CHEMICAL CHARACTERISTICS OF AN ACCEPTABLE DILUTION WATER
C.16. HONEYBEES — ACUTE ORAL TOXICITY TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. VALIDITY OF THE TEST
1.5. DESCRIPTION OF THE TEST METHOD
1.5.1. Collection of bees
1.5.2. Housing and feeding conditions
1.5.3. Preparation of bees
1.5.4. Preparation of doses
1.6. PROCEDURE
1.6.1. Test and control groups
1.6.2. Toxic standard
1.6.3. Exposure
1.6.3.1. Administration of doses
1.6.3.2. Duration
1.6.4. Observations
1.6.5. Limit test
2. DATA AND REPORTING
2.1. DATA
2.2. TEST REPORT
2.2.1. Test substance:
2.2.2. Test species:
2.2.3. Test conditions:
2.2.4. Results:
3. REFERENCES
C.17. HONEYBEES — ACUTE CONTACT TOXICITY TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. VALIDITY OF THE TEST
1.5. DESCRIPTION OF THE TEST METHOD
1.5.1. Collection of bees
1.5.2. Housing and feeding conditions
1.5.3. Preparation of bees
1.5.4. Preparation of doses
1.6. PROCEDURE
1.6.1. Test and control groups
1.6.2. Toxic standard
1.6.3. Exposure
1.6.3.1. Administration of doses
1.6.3.2. Duration
1.6.4. Observations
1.6.5. Limit test
2. DATA AND REPORTING
2.1. DATA
2.2. TEST REPORT
2.2.1. Test substance:
2.2.2. Test species:
2.2.3. Test conditions:
2.2.4. Results:
3. REFERENCES
C.18. ADSORPTION/DESORPTION USING A BATCH EQUILIBRIUM METHOD
1. METHOD
1.1. INTRODUCTION
1.2. SCOPE
1.3. DEFINITIONS AND UNITS
1.4. PRINCIPLE OF THE TEST METHOD
1.5. INFORMATION ON THE TEST SUBSTANCE
1.6. APPLICABILITY OF THE TEST
1.7. DESCRIPTION OF THE METHOD
1.7.1. Apparatus and chemical reagents
1.7.2. Characterisation and selection of soils
1.7.3. Collection and storage of soil samples
1.7.3.1. Collection
1.7.3.2. Storage
1.7.3.3. Handling and preparation of soil samples for the test
1.7.4. Preparation of the test substance for application to soil
1.8. PREREQUISITES FOR PERFORMING THE ADSORPTION/DESORPTION TEST
1.8.1. The analytical method
1.8.2. The selection of optimal soil/solution ratios
1.9. PERFORMANCE OF THE TEST
1.9.1. Test conditions
1.9.2. Tier 1 — Preliminary study
1.9.2.1. Selection of optimal soil/solution ratios
1.9.2.2. Determination of adsorption equilibration time and of the amount of test substance adsorbed at equilibrium
1.9.2.3. Adsorption on the surface of the test vessel and stability of the test substance
1.9.3. Tier 2 — Adsorption kinetics at one concentration of the test substance
1.9.4. Tier 3 — Adsorption isotherms and desorption kinetics/desorption isotherms
1.9.4.1.
Adsorption isotherms
1.9.4.2.
Desorption kinetics
1.9.4.3.
Desorption isotherms
2. DATA AND REPORTING
2.1. ADSORPTION
2.1.1. Adsorption isotherms
2.1.2. Mass balance
2.2. DESORPTION
2.2.1. Desorption isotherms
2.2.2. Test report
3. REFERENCES
Appendix 1
Testing scheme
Appendix 2
INFLUENCE OF ACCURACY OF ANALYTICAL METHOD AND CONCENTRATION CHANGE ON ACCURACY OF ADSORPTION RESULTS
Appendix 3
ESTIMATION TECHNIQUES FOR K
D
Appendix 4
CALCULATIONS FOR DEFINING THE CENTRIFUGATION CONDITIONS
Fig. 1a.
Fig. 1b.
Appendix 5
CALCULATION OF ADSORPTION A (%) AND DESORPTION D (%)
ADSORPTION A (A%)
a) Parallel method
b) Serial method
DESORPTION D (A %)
(a) Parallel method
(b) Serial method
Appendix 6
ADSORPTION-DESORPTION IN SOILS: DATA REPORTING SHEETS
Suitability of the analytical method
Adsorption test: test samples
Adsorption test: blanks and control
Mass balance
Adsorption isotherms
Desorption test
C.19. ESTIMATION OF THE ADSORPTION COEFFICIENT (
K
OC
) ON SOIL AND ON SEWAGE SLUDGE USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. APPLICABILITY OF THE TEST
1.6. QUALITY CRITERIA
1.6.1. Accuracy
1.6.2. Repeatability
1.6.3. Reproducibility
1.7. DESCRIPTION OF THE TEST METHOD
1.7.1. Preliminary Estimation of the Adsorption Coefficient
1.7.2. Apparatus
1.7.3. Mobile phases
1.7.4. Solutes
1.8. PERFORMANCE OF THE TEST
1.8.1. Test condition
1.8.2. Determination of dead time to
1.8.2.1. Determination of the dead time to by means of a homologous series
1.8.2.2. Determination of the dead time to by inert substances which are not retained by the column
1.8.3. Determination of the retention times tR
1.8.4. Evaluation
2. DATA AND REPORTING
3. REFERENCES
Appendix
C.20
DAPHNIA MAGNA
REPRODUCTION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS AND UNITS
1.3. PRINCIPLE OF THE TEST METHOD
1.4. INFORMATION ON THE TEST SUBSTANCE
1.5. VALIDITY OF THE TEST
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Apparatus
1.6.2. Test organism
1.6.3. Test medium
1.6.4. Test solutions
1.7. TEST DESIGN
1.8. PROCEDURE
1.8.1. Conditions of exposure
1.8.1.1. Duration
1.8.1.2. Loading
1.8.1.3. Number of animals
1.8.1.4. Feeding
1.8.1.5. Light
1.8.1.6. Temperature
1.8.1.7. Aeration
1.8.2. Test concentration
1.8.3. Controls
1.8.4. Test medium renewal
1.8.5. Observations
1.8.6. Offspring
1.8.7. Mortality
1.8.8. Other parameters
1.8.9. Frequency of analytical determinations and measurements
2. DATA AND REPORTING
2.1. TREATMENT OF RESULTS
2.2. TEST REPORT
2.2.1. Test substance:
2.2.2. Test species:
2.2.3. Test conditions:
2.2.4. Results:
3. REFERENCES
Appendix 1
PREPARATION OF FULLY DEFINED ELENDT M7 AND M4 MEDIA
Acclimation to Elendt M7 and M4 media
PREPARATION
Trace elements
M4 and M7 media
Appendix 2
TOTAL ORGANIC CARBON (TOC) ANALYSIS AND PRODUCTION OF A NOMOGRAPH FOR TOC CONTENT OF ALGAL FEED
Appendix 3
EXAMPLE DATA SHEET RECORDING MEDIUM RENEWAL, PHISICAL/CHEMICAL MONITORING DATA, FEEDING,
DAPHNIA
REPRODUCTION AND ADULT MORTALITY
Appendix 4
EXAMPLE DATA SHEET FOR RECORDING RESULTS OF CHEMICAL ANALYSIS
(a) Measured concentrations
(b) Measured concentrations as a percentage of nominal
Appendix 5
CALCULATION OF A TIME-WEIGHTED MEAN
Time-weighted mean
Figure 1: Example of time-weighted mean
C.21. SOIL MICROORGANISMS: NITROGEN TRANSFORMATION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. VALIDITY OF THE TEST
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Apparatus
1.6.2. Selection and number of soils
1.6.3. Collection and storage of soil samples
1.6.3.1. Collection
1.6.3.2. Storage
1.6.4. Handling and preparation of soil for the test
1.6.4.1. Pre-incubation
1.6.4.2. Physical-chemical characteristics
1.6.4.3. Amendment with organic substrate
1.6.5. Preparation of the test substance for the application to soil
1.6.6. Test concentrations
1.7. PERFORMANCE OF THE TEST
1.7.1. Conditions of exposure
1.7.1.1. Treatment and control
1.7.1.2. Incubation of soil samples
1.7.1.3. Test conditions and duration
1.7.2. Sampling and analysis of soils
1.7.2.1. Soil sampling schedule
1.7.2.2. Analysis of soil samples
2. DATA
2.1. TREATMENT OF RESULTS
2.2. INTERPRETATION OF RESULTS
3. REPORTING
4. REFERENCES
C.22. SOIL MICROORGANISMS: CARBON TRANSFORMATION TEST
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. PRINCIPLE OF THE TEST METHOD
1.5. VALIDITY OF THE TEST
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Apparatus
1.6.2. Selection and number of soils
1.6.3. Collection and storage of soil samples
1.6.3.1. Collection
1.6.3.2. Storage
1.6.4. Handling and preparation of soil for the test
1.6.4.1. Pre-incubation
1.6.4.2. Physical-chemical characteristics
1.6.5. Preparation of the test substance for the application to soil
1.6.6. Test concentrations
1.7. PERFORMANCE OF THE TEST
1.7.1. Conditions of exposure
1.7.1.1. Treatment and control
1.7.1.2. Incubation of soil samples
1.7.1.3. Test conditions and duration
1.7.2. Sampling and analysis of soils
1.7.2.1. Soil sampling schedule
1.7.2.2. Measurement of glucose-induced respiration rates
2. DATA
2.1. TREATMENT OF RESULTS
2.2. INTERPRETATION OF RESULTS
3. REPORTING
TEST REPORT
4. REFERENCES
C.23. AEROBIC AND ANAEROBIC TRANSFORMATION IN SOIL
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. APPLICABILITY OF THE TEST
1.5. INFORMATION ON THE SUBSTANCE
1.6. PRINCIPLE OF THE TEST METHOD
1.7. QUALITY CRITERIA
1.7.1. Recovery
1.7.2. Repeatability and sensitivity of analytical method
1.7.3. Accuracy of transformation data
1.8. DESCRIPTION OF THE METHOD
1.8.1. Equipment and chemical reagents
1.8.2. Test substance application
1.8.3. Soils
1.8.3.1. Soil selection
1.8.3.2. Collection, handling, and storage of soils
1.9. PERFORMANCE OF THE TEST
1.9.1. Test conditions
1.9.1.1. Test temperature
1.9.1.2. Moisture content
1.9.1.3. Aerobic incubation conditions
1.9.1.4. Sterile aerobic conditions
1.9.1.5. Anaerobic incubation conditions
1.9.1.6. Paddy incubation conditions
1.9.1.7. Test duration
1.9.2. Performance of the test
1.9.3. Sampling and measurement
1.9.4. Optional tests
2. DATA
2.1. TREATMENT OF RESULTS
2.2. EVALUATION AND INTERPRETATION OF RESULTS
3. REPORTING
TEST REPORT
4. REFERENCES
Appendix 1
WATER TENSION, FIELD CAPACITY (FC) AND WATER HOLDING CAPACITY (WHC)(47)
Appendix 2
SOIL MOISTURE CONTENTS (g water per 100 g dry soil) OF VARIOUS SOIL TYPES FROM VARIOUS COUNTRIES
Appendix 3
Figure 1
Example of a flow-through apparatus to study transformation of chemicals in soil
(53) (54)
Figure 2
Example of a biometer-type flask for studying the transformation of chemicals in soil
(55)
C.24. AEROBIC AND ANAEROBIC TRANSFORMATION IN AQUATIC SEDIMENT SYSTEMS
1. METHOD
1.1. INTRODUCTION
1.2. DEFINITIONS
1.3. REFERENCE SUBSTANCES
1.4. INFORMATION ON THE TEST SUBSTANCE
1.5. PRINCIPLE OF THE TEST METHOD
1.6. APPLICABILITY OF THE TEST
1.7. QUALITY CRITERIA
1.7.1. Recovery
1.7.2. Repeatability and sensitivity of analytical method
1.7.3. Accuracy of transformation data
1.8. DESCRIPTION OF THE METHOD
1.8.1. Test system and apparatus
1.8.2. Selection and number of aquatic sediments
1.8.2.1. Sediment selection
1.8.2.2. Characterisation of water-sediment samples
1.8.3. Collection, handling and storage
1.8.3.1. Collection
1.8.3.2. Handling
1.8.3.3. Storage
1.8.4. Preparation of the sediment/water samples for the test
1.9. PERFORMANCE OF THE TEST
1.9.1. Test conditions
1.9.2. Treatment and application of test substance
1.9.3. Test duration and sampling
1.9.4. Optional preliminary test
1.9.5. Measurements and analysis
2. DATA
2.1. TREATMENT OF RESULTS
3. REPORTING
3.1. TEST REPORT
4. REFERENCES
Appendix 1
GUIDANCE ON THE AEROBIC AND THE ANAEROBIC TEST SYSTEMS
Aerobic test system
Anaerobic test system
Appendix 2
EXAMPLE OF A GAS FLOW-THROUGH APPARATUS
Appendix 3
EXAMPLE OF A BIOMETER APPARATUS
Appendix 4
EXAMPLE CALCULATION FOR APPLICATION DOSE TO TEST VESSELS

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