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Reaction mass of ammonium;potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetate;magnesium, ammonium;potassium;sodium;2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetate;magnesium and ammonium;potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(2-hydroxyethyl)amino]acetate;magnesium
EC number: 902-533-2 | CAS number: -
- 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
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- Additional physico-chemical information
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- Endpoint summary
- Stability
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- 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
Based on in-vitro guideline studies, the substance does not cause skin or eye irritation.
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
- Version / remarks:
- 29 July 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EU Method B.40bis
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Source strain:
- not specified
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS, Cell Systems
- Tissue batch number(s): Batch No.100-AG1428-1)
- Production date: 02 October 2017
- Shipping date: 02 October 2017
- Delivery date: 03 October 2017
- Date of initiation of testing: 04 October 2017
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2,
- Temperature of post-treatment incubation (if applicable): for 2 hours 55 minutes between 36.5°C and 38°C, 5% CO2..
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 3 minutes and 55 minutes after the test item application, the human epidermis was washed 20 times with 1 mL of DPBS (DPBS – Dutscher, Batch No. 1310817).
- Observable damage in the tissue due to washing:
The rinsed tissues were checked for any coloration and noted to be whitish, comparable coloration to that of the negative control tissues.
- Modifications to validated SOP: not specified
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time:
The skin samples were placed in 300 μL of a MTT solution at 1 mg/mL for 2 hours and 55 minutes between 36.5°C and 38.0°C, 5% CO2.
- Spectrophotometer: not specified
- Wavelength: 570 nm
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function: 55.00%
- Morphology: sufficient cornified (5) and vital (4) cell layers
- Contamination: no indication of HIV1, HBV, HCV, bacteria, yeast or mycoplasma found.
- Reproducibility: not specified
NUMBER OF REPLICATE TISSUES: 2
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
no interference
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if Viability measured after exposure time points (t=3 and 60 minutes) < 50% after 3 min exposure
- The test substance is considered to be corrosive to skin if Viability measured after exposure time points (t=3 and 60 minutes) ≥ 50% after 3 min exposure AND < 15% after 60 min exposure
- The test substance is considered to be non-corrosive to skin if Viability measured after exposure time points (t=3 and 60 minutes) ≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: not applicable - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
CONTROLS
In the same experimental conditions, a positive control (50 μL of 8N KOH – Sigma, Batch No. SLBD3295V) and a negative control (50 μL of distilled water– Prochilab, Batch No. 1007123) were carried out. - Duration of treatment / exposure:
- The test item was applied, at the dose of 25 mg, during 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2., to the epidermal surface of 2 living human skin models previously moistened with 25 μL of distilled water.
- Duration of post-treatment incubation (if applicable):
- The skin samples were placed in 300 μL of a MTT solution at 1 mg/mL for 2 hours and 55 minutes between 36.5°C and 38.0°C, 5% CO2.
- Number of replicates:
- 2
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Replicate 1
- Value:
- 93.1
- Vehicle controls validity:
- not examined
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Replicate 2
- Value:
- 87.12
- Vehicle controls validity:
- not examined
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- 3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 93.10% and 87.12%, versus 57.77% and 0.62%, respectively, with the positive control item (potassium hydroxide 8N).
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA does not have to be classified in Category 1 “Corrosive”.
The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required. - Executive summary:
The aim of the study was to evaluate the possible corrosive effects of the test item after topical administration onin vitrohuman reconstituted epidermis (epiCS®, CellSystems®).
The test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA was applied as supplied, at the dose of 25 mg, to 2 living Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 55 minutes. The application was followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The experimental protocol was established in accordance with the O.E.C.D. Test Guideline No. 431 dated 28 July 2015 and the method B.40bis of the Council regulation No. 440/2008.
3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 93.10% and 87.12%, versus 57.77% and 0.62%, respectively, with the positive control item (potassium hydroxide 8N).
In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item Reaction mixture of MgEDTA, MgDTPA andMgHEEDTA does not have to be classified in Category 1 “Corrosive”.
The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- adopted 28 July 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Version / remarks:
- 23 July 2009
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- foreskin from a single donor
- Source strain:
- not specified
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin SA, RHE/S/17
- Tissue batch number(s): Batch No. 17-RHE-108
- Production date: 17 October 2017
- Shipping date: 17 October 2017
- Delivery date: 17 October 2017
- Date of initiation of testing: 17 October 2017
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 42 minutes after the test item application, the human epidermis were washed with 25 x 1 mL of DPBS (Dutscher - Batch No. 1310817).
- Observable damage in the tissue due to washing: The rinsed tissues were checked for any coloration and noted to be whitish, comparable to negative control tissues.
- Modifications to validated SOP: not specified
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Wavelength: 570 nm
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: CV= 5.6%
- Barrier function: not determined, 6.5 cell layers
- Morphology: no abnormalities, well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum
- Contamination: on blood of the same donor, the absence of HIV1 and 2 antibodies, hepatitis C antibodies and hepatitis B antigen HB sp was confirmed. On epidermal cells of the smae donor, th absence of mycoplasma was confirmed.
- Reproducibility: not specified
NUMBER OF REPLICATE TISSUES: 3
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
no MTT interference
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-corrosive to skin if if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.
The test item is identified as requiring classification and labelling according to UN GHS (Category 2):
if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is “non corrosive”.
The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1):
if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and in absence of information on a skin corrosion test.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: not applicalble - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
The test item was applied as supplied, during 42 minutes at room temperature at the dose of 16 mg to the epidermal surface of 3 living Reconstructed Human epidermis previously moistened with 10 μL of distilled water.
POSITIVE/NEGATIVE CONTROL
In the same experimental conditions, a positive control (5% SDS), and a negative control (DPBS – Dutscher - Batch No. 1310817) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS (SIGMA Batch No. STBG6142V) in a 10 mL volumetric flask qsp 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment.
To ensure a good contact with the epidermis, during all the treatment period, all liquid items were recovered with a nylon mesh provided by Episkin SA. - Duration of treatment / exposure:
- 42 minutes
- Duration of post-treatment incubation (if applicable):
- 41 hours 30 minutes
- Number of replicates:
- 3
- Irritation / corrosion parameter:
- % tissue viability
- Value:
- 89.8
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- The mean corrected percent viability of the treated tissues was 89.8%, versus 2.3% in the positive control (5% Sodium Dodecyl Sulfate).
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In accordance with the Regulation EC No. 1272/2008, the test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category.
No hazard statement or signal word is required. - Executive summary:
The aim was to evaluate the possible irritating effects of the test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA after topical application onin vitrohuman reconstructed epidermis (SkinEthic RHE®model).
The test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA was applied as supplied, during 42 minutes, at the dose of 16 mg, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) previously moistened with 10 μL of distilled water. The application was followed by a rinse with 25 mL of DPBS and a 42 hours and 30 minutes post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The experimental protocol was established in accordance with O.E.C.D. Test Guideline No. 439 adopted 28 July 2015 and the Test method B.46 of Council regulation No. 761/2009 dated 23 July 2009 (EU Journal L220) - ATP Council regulation No. 440/2008 of 30 May 2008 (E.U. Journal L142).
The mean corrected percent viability of the treated tissues was 89.8%, versus 2.3% in the positive control (5% Sodium Dodecyl Sulfate).
In accordance with the Regulation EC No. 1272/2008, the test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category.
No hazard statement or signal word is required.
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 405 (Acute Eye Irritation / Corrosion)
- Version / remarks:
- October 2nd, 2012.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or tissues and environmental conditions:
- TEST ANIMALS
- Source: Hypharm (F-49450 La Renaudiere)
- Age at study initiation: 11 weeks
- Weight at study initiation: 2.05 - 2.43 kg
- Housing:
Each animal was kept in an individual box installed in conventional air conditioned animal husbanding:
- Diet (e.g. ad libitum):
Drinking water (tap-water from public distribution system) and foodstuff (ENVIGO – 2030C) were supplied ad libitum.
Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas – Eurofins (FRANCE).
- Water (e.g. ad libitum):
Drinking water (tap-water from public distribution system) and foodstuff (ENVIGO – 2030C) were supplied ad libitum.
Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas – Eurofins (FRANCE).
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
The temperature and relative humidity of the main test were controlled to remain within target ranges of 17°C to 23°C and 30% to 70%, respectively.
The rate of air exchange was at least ten changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (07.00 to 19.00) and twelve hours darkness.
- Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent no treatment
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1g
- Duration of treatment / exposure:
- 0.1 g of the test item was instilled, as supplied, into the conjunctival sac of one eye after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second in order to prevent loss of the test item.
- Observation period (in vivo):
Ocular examinations were performed on both right and left eyes 1 hour, 24, 48 and 72 hours following treatment- Number of animals or in vitro replicates:
- 3
- Details on study design:
- REMOVAL OF TEST SUBSTANCE
- Washing (if done): no washing
SCORING SYSTEM:
See "any other information on materials and methods"
TOOL USED TO ASSESS SCORE: not specified - Irritation parameter:
- chemosis score
- Basis:
- animal #1
- Time point:
- 24/48/72 h
- Score:
- 0.3
- Max. score:
- 1
- Reversibility:
- fully reversible within: 48 h
- Irritation parameter:
- chemosis score
- Basis:
- animal #2
- Time point:
- 24/48/72 h
- Score:
- 0.7
- Max. score:
- 1
- Reversibility:
- fully reversible within: 72 h
- Irritation parameter:
- chemosis score
- Basis:
- animal #3
- Time point:
- 24/48/72 h
- Score:
- 0.3
- Max. score:
- 1
- Reversibility:
- not fully reversible within: 48 h
- Irritation parameter:
- conjunctivae score
- Remarks:
- redness
- Basis:
- animal #1
- Time point:
- 24/48/72 h
- Score:
- 1.3
- Max. score:
- 2
- Reversibility:
- not fully reversible within: 72 hours
- Irritation parameter:
- conjunctivae score
- Remarks:
- redness
- Basis:
- animal #2
- Time point:
- 24/48/72 h
- Score:
- 1
- Max. score:
- 2
- Reversibility:
- fully reversible within: 72 hours
- Irritation parameter:
- conjunctivae score
- Remarks:
- redness
- Basis:
- animal #3
- Time point:
- 24/48/72 h
- Score:
- 1.7
- Max. score:
- 2
- Reversibility:
- not fully reversible within: 72 hours
- Irritation parameter:
- iris score
- Remarks:
- lesion
- Basis:
- animal #1
- Time point:
- 24/48/72 h
- Score:
- 0
- Max. score:
- 0
- Irritation parameter:
- iris score
- Remarks:
- lesion
- Basis:
- animal #2
- Time point:
- 24/48/72 h
- Score:
- 0
- Max. score:
- 0
- Irritation parameter:
- iris score
- Remarks:
- lesion
- Basis:
- animal #3
- Time point:
- 24/48/72 h
- Score:
- 0
- Max. score:
- 0
- Irritation parameter:
- cornea opacity score
- Basis:
- animal #1
- Time point:
- 24/48/72 h
- Score:
- 1
- Max. score:
- 1
- Reversibility:
- not fully reversible within: 72 h
- Irritation parameter:
- cornea opacity score
- Basis:
- animal #2
- Time point:
- 24/48/72 h
- Score:
- 0
- Max. score:
- 0
- Irritation parameter:
- cornea opacity score
- Basis:
- animal #3
- Time point:
- 24/48/72 h
- Score:
- 0
- Max. score:
- 0
- Irritant / corrosive response data:
The ocular reactions observed during the study have been slight to moderate and totally reversible:
- at the conjunctivae level: a moderate redness noted 1 hour after the test item instillation and totally reversible between days 3 and 7. This reaction was associated with a moderate chemosis
noted 1 hour after the test item instillation and totally reversible between days 2 and 3.
- at the iris level: an injection noted 1 hour after the test item instillation in one animal and totally reversible on day 1.
- at the corneal level: a slight opacity noted 24 hours after instillation in one animal and totally reversible on day 7.- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The results obtained, under these experimental conditions, enable to conclude that the test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA does not have to be classified in accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures.
No symbol and warning label are required. - Executive summary:
The test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA was instilled, as supplied, into the eye of three New Zealand rabbits at the dose of 0.1 g. The experimental protocol was established on the basis of the official method as defined in the O.E.C.D. Test Guideline No. 405 dated October 2nd, 2012.
The ocular reactions observed during the study have been slight to moderate and totally reversible:
-
- at the conjunctivae level: a moderate redness noted 1 hour after the test item instillation and totally reversible between days 3 and 7. This reaction was associated with a moderate chemosis noted 1
hour after the test item instillation and totally reversible between days 2 and 3.
-
- at the iris level: an injection noted 1 hour after the test item instillation in one animal and totally
reversible on day 1.
-
- at the corneal level: a slight opacity noted 24 hours after instillation in one animal and totally
reversible on day 7.
In conclusion, the results obtained, under these experimental conditions, enable to conclude that the test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA does not have to be classified in accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures.
No symbol and warning label are required.
-
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
- Version / remarks:
- adopted 26 July 2013
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
- Version / remarks:
- 08 December 2010
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Species:
- chicken
- Details on test animals or tissues and environmental conditions:
- SOURCE OF COLLECTED EYES
- Source:
The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight):
The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): see below
- Time interval prior to initiating testing:
Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 04 October 2017 at 8:25 am.
Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.
The eyes were enucleated at Phycher on 04 October 2017 at 09:55 am.
- indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: no - Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Immediately following the zero reference measurements, three eyes (in their holder) were removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied, as supplied during 10 seconds, to the cornea such that the entire surface of the cornea was evenly covered with the test item.
- Duration of treatment / exposure:
- single application
- Duration of post- treatment incubation (in vitro):
- Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.
- Number of animals or in vitro replicates:
- three eyes
- Details on study design:
- SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 31.4°C and 32.3°C.
After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.
EQUILIBRATION AND BASELINE RECORDINGS
Once all eyes had been examined and approved (see table in appendix 4), the eyes were incubated between 45 and 57 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.
NUMBER OF REPLICATES
3
NEGATIVE CONTROL USED
physiological saline – 1 replicate
SOLVENT CONTROL USED (if applicable)
not applicable
POSITIVE CONTROL USED
sodium hydroxide –- 3 replicates
APPLICATION DOSE AND EXPOSURE TIME
Immediately following the zero reference measurements, three eyes (in their holder) were removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied, as supplied during 10 seconds, to the cornea such that the entire surface of the cornea was evenly covered with the test item.
OBSERVATION PERIOD
Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.
REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Tthe eyes were rinsed twice with 10 mL of physiological saline at ambient temperature. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.
- Indicate any deviation from test procedure in the Guideline
No deviation was registered during the study.
METHODS FOR MEASURED ENDPOINTS:
See any other information on materials and methods.
SCORING SYSTEM:
See any other information on materials and methods.
DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
See any other information on materials and methods. - Irritation parameter:
- cornea opacity score
- Value:
- <= 2
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation parameter:
- fluorescein retention score
- Value:
- 3
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation parameter:
- percent corneal swelling
- Value:
- <= 3
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- The combination of the three endpoints for the test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA was 1 x IV, 1 x III, 1 x I.
The combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.
The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected. - Interpretation of results:
- study cannot be used for classification
- Conclusions:
- In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, the test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing (in vitro and/or in vivo) are required to establish a definitive classification.
- Executive summary:
The aim of the study was to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.
The test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA was applied, as supplied, at the dose of 30 mg, to 3 enucleated chicken eyes, during 10 seconds. Then eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose. The experimental protocol was established in accordance with theO.E.C.D. Test Guideline No. 438 adopted 26 July 2013 and the test method B.48–Commission Regulation (EU) No. 1152/2010 dated 08 December 2010 (EU Journal L324) - ATP Council regulation No. 440/2008 of 30 May 2008 (E.U. Journal L142).
The ocular reactions observed in eyes treated with the test item were:
-
- maximal mean score of corneal opacity: 2.0, corresponding to ICE class III;
-
- mean score of fluorescein retention: 3.0, corresponding to ICE class IV;
-
- maximal mean corneal swelling: 3%, corresponding to ICE class I.
The combination of the three endpoints for the test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA was 1 x IV, 1 x III, 1 x I.
The combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV.Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.
The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.
In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, the test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing (in vitro and/or in vivo) are required to establish a definitive classification.
-
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
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