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EC number: 934-512-9 | 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
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Eye irritation
Administrative data
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From September 20, 2017 to September 22, 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
- Deviations:
- yes
- Remarks:
- Deviations were considered to have not affected the integrity or validity of the study
- Qualifier:
- according to guideline
- Guideline:
- EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
- Deviations:
- yes
- Remarks:
- Deviations were considered to have not affected the integrity or validity of the study
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Potassium lauroyl wheat amino acids
- Molecular formula:
- Representative molecular formula for the major constituents/amino acids: C17H30N1O3K1 (potassium lauroyl proline) C14H26N1O3K1 (potassium lauroyl glycine) C18H34N1O3K1 (potassium lauroyl leucine) C15H28N1O4K1 (potassium lauroyl serine) C17H30N1O5K1 (potassium lauroyl glutamic acid)
- IUPAC Name:
- Potassium lauroyl wheat amino acids
- Test material form:
- other: Semi-solid
Constituent 1
Test animals / tissue source
- Species:
- cattle
- Strain:
- not specified
- Details on test animals or tissues and environmental conditions:
- Source of Bovine Eyes
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were refrigerated on arrival and used within 24 h of receipt.
Test system
- Vehicle:
- physiological saline
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- 0.75 mL test substance or reference substances (Positive control: neat Ethanol, Negative control: Sodium chloride 0.9% w/v)
- Duration of treatment / exposure:
- At 32 ± 1 ºC for 10 minutes
- Duration of post- treatment incubation (in vitro):
- At 32 ± 1 ºC for 90 minutes for permeablity assessment
- Number of animals or in vitro replicates:
- Three corneas to each test substance and reference substances
- Details on study design:
- Preparation of corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
Selection of corneas and opacity reading
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test substance and three corneas to the positive control substance.
Treatment of corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test substance or control substances were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the substance over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1ºC for 10 minutes. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed. The negative and positive control data was shared with Envigo study number PQ52DX and HN60FW.
Application of sodium fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.
Permeability determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.360 µL of media representing each cornea was dispensed into the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.
Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin. In this study histopathology was not required.
Data evaluation
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
Opacity measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
Permeability measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
In Vitro irritancy score
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test substance induced a response through only one of the two endpoints.
Visual observation
The condition of the cornea was visually assessed post treatment.
The negative and positive control data was shared with Envigo study number PQ52DX and HN60FW.
Results and discussion
In vitro
Resultsopen allclose all
- Irritation parameter:
- in vitro irritation score
- Run / experiment:
- Test substance
- Value:
- ca. 19.6
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: IVIS is within the range: ≥3-≤55; therefore no predication can be made
- Irritation parameter:
- in vitro irritation score
- Run / experiment:
- Positive control
- Value:
- ca. 45.6
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Irritation parameter:
- in vitro irritation score
- Run / experiment:
- Negative control
- Value:
- ca. 0.7
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- The positive control In Vitro Irritancy Score was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied.
Any other information on results incl. tables
Results
Corneal Opacity and Permeability measurement
Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in following table 1:
Treatment |
Cornea Number |
Opacity |
Permeability (OD) |
In Vitro Irritancy Score |
|||||
Pre-Treatment |
Post-Treatment |
Post Incubation |
Post-Incubation - Pre‑Treatment |
Corrected Value |
|
Corrected Value |
|||
Negative Control* |
1 |
3 |
3 |
3 |
0 |
|
0.000 |
|
|
2 |
5 |
6 |
6 |
1 |
|
0.002 |
|
|
|
3 |
3 |
3 |
4 |
1 |
|
0.001 |
|
|
|
Mean |
|
|
|
0.7 |
|
0.001 |
|
0.7 |
|
Positive Control* |
4 |
4 |
36 |
34 |
30 |
29.3 |
0.659 |
0.658 |
|
5 |
4 |
35 |
35 |
31 |
30.3 |
1.276 |
1.275 |
|
|
6 |
2 |
35 |
35 |
33 |
32.3 |
1.061 |
1.060 |
|
|
Mean |
|
|
|
|
30.7 |
|
0.998 |
45.6 |
|
Test Substance |
10 |
2 |
16 |
17 |
15 |
14.3 |
0.265 |
0.264 |
|
11 |
6 |
22 |
20 |
14 |
13.3 |
0.402 |
0.401 |
|
|
12 |
3 |
13 |
16 |
13 |
12.3 |
0.588 |
0.587 |
|
|
Mean |
|
|
|
|
13.3 |
|
0.417 |
19.6 |
OD= Optical density
* = The negative and positive control data was shared with Envigo study number PQ52DX and HN60FW.
Corneal Epithelium Condition
The condition of each cornea is given in below table :
Treatment |
Cornea Number |
Observation |
|
Post Treatment |
Post Incubation |
||
Negative Control* |
1 |
Clear |
Clear |
2 |
Clear |
Clear |
|
3 |
Clear |
Clear |
|
Positive Control* |
4 |
Cloudy |
Cloudy |
5 |
Cloudy |
Cloudy |
|
6 |
Cloudy |
Cloudy |
|
Test Substance |
10 |
Partly Cloudy |
Cloudy |
11 |
Partly Cloudy |
Cloudy |
|
12 |
Partly Cloudy |
Cloudy |
*= Control data shared with Envigo study number PQ52DX and HN60FW
The corneas treated with the test substance were partly cloudy post treatment and post incubation. The corneas treated with the negative control substance were clear post treatment and post incubation. The corneas treated with the positive control substance were cloudy post treatment and post incubation.
In Vitro Irritancy Score
The In Vitro irritancy scores are summarized as follows:
Treatment |
In Vitro Irritancy Score |
Test Substance |
19.6 |
Negative Control |
0.7 |
Positive Control |
45.6 |
Criteria for an Acceptable Test
The positive control In Vitro Irritancy Score was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied.
Conclusion
No prediction of eye irritation can be made.
Applicant's summary and conclusion
- Interpretation of results:
- other: no prediction could be made
- Conclusions:
- Under the study conditions, no prediction of eye irritation could be made for the test substance.
- Executive summary:
An in vitro study was conducted to determine the eye irritation potential of the test substance, 'potassium lauroyl wheat amino acids' (active: 69.2%), using the Bovine corneal Opacity Test (BCOP) method, according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. Preparation, selection and opacity reading of the corneas were performed as per the guideline. Prepared corneas in triplicates were treated with each, test substance (undiluted), negative control (sodium chloride 0.9% w/v) and positive control (neat ethanol) substances at 32 ± 1ºC for 10 minutes. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete Eagle’sMinimum Essential Medium (EMEM) containing phenol red before a final rinse with complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed. The negative and positive control data was shared with Envigo study number PQ52DX and HN60FW. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1ºC for 90 minutes. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The IVIS of the test substance was determined to be 19.6 which is well below the corrosive limit of 55 and above the non-corrosive limit of 3. The positive control IVIS was within the range of 31.6 to 58.7; therefore acceptance criterion was satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077; therefore the negative control acceptance criteria were satisfied. T The quality criteria required for acceptance of results in the test were satisfied. Under the study conditions, no prediction of eye irritation could be made for the test substance (Envigo, 2018).
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