Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 850-366-8 | CAS number: 98969-19-8
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Description of key information
Skin irritation, in vitro: non-irritating (15 minutes exposure/42hour incubation: mean relative viability = 82 ± 1.3 %), EPISKIN, OECD TG 439, 2020
Eye irritation: non-irritating; ICE class I x 3, OECD TG 438, 2020
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 10-03-2020 to 23-03-2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Guideline study performed under GLP with minor deviations. All relevant validity criteria were considered to be met.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Deviations:
- yes
- Remarks:
- Minor deviation in environmental conditions (humidity minimum 72%) ; 25 µL test item, negative control, positive controls were applied, respectively rather than 10 µL. These protocol deviations were not considered to affect the reliability of the study.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Deviations:
- yes
- Remarks:
- Minor deviation in environmental conditions (humidity minimum 72%) ; 25 µL test item, negative control, positive controls were applied, respectively rather than 10 µL. These protocol deviations were not considered to affect the reliability of the study.
- GLP compliance:
- yes
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- foreskin from a single donor
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM Small Model
- Tissue batch number(s): Lot no.: 20-EKIN-012
- Production date: Not reported.
- Shipping date: Not reported. Although the CoA for the tissues QA is provided in the full study report.
- Delivery date: 10-03-2020 ; day prior to test initiation (ca. 24 hours)
- Date of initiation of testing: ca. 10-03-2020
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.0 °C, 5 ± 0.5% CO2 (actual: 34.3 – 37.0°C)
- Temperature of post-treatment incubation (if applicable): 37 ± 1.0 °C, 5 ± 0.5% CO2; for 42 hours. For further information see below.
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. For the cell viability measurements: cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labelled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for approximately 68 hours. The amount of extracted formazan was determined spectrophotometrically.
- Observable damage in the tissue due to washing: Not applicable.
- Modifications to validated SOP: Not applicable.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3.0 mg/mL MTT concentrated diluted (x10) to 0.3 mg/mL MTT solution prepared in assay medium
- Incubation time: For the cell viability measurements: cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labelled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for approximately 72 hours. The amount of extracted formazan was determined spectrophotometrically.
- Spectrophotometer: M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: Without reference filter
- Filter bandwidth: No reported.
- Linear OD range of spectrophotometer: Not reported.
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Within NC and PC historic control ranges presented within the report.
- Barrier function: See above.
- Morphology: See above.
- Contamination: Not applicable.
- Reproducibility: The relative mean tissue viability for the positive control treated tissues was 8.4% relative to the negative control following the 15-minute exposure/42-hour incubation period. With standard deviation of 1.9%. The positive control acceptance criterion was therefore satisfied. The negative control triplicate treated tissues mean OD570 was 1.0336 and the standard deviation of viability was 16%. The acceptance criterion was therefore satisfied. The test item triplicate treated tissues viability standard deviation was 1.3%. The mean OD570 for both the negative and positive controls were within the historical control ranges.
- Other: As an additional quality control step, the results of the assay are compared to the historical ranges for negative and positive controls generated by the testing laboratory to confirm the functioning of the test system.
NUMBER OF REPLICATE TISSUES: Three (3), triplicate
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: In the pretest for colour interference: the solution containing the test item was colourless. It was therefore unnecessary to run colour correction tissues. In the pre-test for direct MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT. It was unnecessary to run killed tissues.
- Procedure used to prepare the killed tissues (if applicable): Not applicable.
- N. of replicates : Not applicable.
- Method of calculation used: Not applicable.
- Other: In the assessment of Colour Interference with the MTT endpoint, the solution containing the test item did not become coloured. This was taken to indicate the test item did not have the potential to cause colour interference.
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One (n=1)
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if e.g. the viability after 15-minutes exposure / 42-hours incubation is less than or equal to 50%,.
- The test substance is considered to be non-irritating to skin if e.g. the viability after 15-minutes exposure / 42-hours incubation is greater than 50%.
- Cut off points in accordance with OECD TG 439 and the GHS and CLP Classification systems.
- Skin irritation is expressed as the remaining cell viability after exposure to the test substance at exposure times 15-minutes / 42-hours incubation. Where necessary, direct MTT reduction, colour interference modifications were completed.
OTHER:
EpiSkin RHE Small Model (Lot no.: 20-EKIN-012). The test consists of topical application of the test item -one on the skin tissue for 15 minutes. After exposure the skin tissue is thoroughly rinsed to remove the test item and transferred to fresh medium. After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect is performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment. The EPISKIN Small Model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Pre-test checks for Direct MTT reduction and Colour Interference were completed.
Preincubation:
On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 22.5 hours at 37°C. Maintenance medium and Assay medium were supplied by a recognised supplier (documented in the full study report).
Application of test item and rinsing:
Test item: Tissues were treated with 25 µL test item for 15 minutes exposure period. Negative control: 25 µL PBS (negative control) was similarly applied. Positive control: 25 µL SDS (5% w/v) was respread after 7 minutes contact time to maintain distribution of the SDS - for the remainder of the contact period. This was not deemed necessary in the negative control or test item definitive test group. Minor deviation : 25 µL test item, negative control, positive controls were applied (ca. 65 µL/cm2), respectively rather than 10 µL (ca. 26 µL/cm2). These protocol deviations were not considered to affect the reliability of the study. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 72 – 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.3 - 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on testing laboratory historical data these deviations are not considered significant. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL
- Concentration (if solution): undiluted
- Other: Minor deviation : 25 µL test item, negative control, positive controls were applied (ca. 65 µL/cm2), respectively rather than 10 µL (ca. 26 µL/cm2). These protocol deviations were not considered to affect the reliability of the study
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution): Not applicable. (Phosphate buffered saline, pre-diluted)
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution): 5% SDS (pre-diluted) - Duration of treatment / exposure:
- 15 ± 0.5 minutes at room temperature, after which the tissues were washed with phosphate buffered saline to remove residual test item.
- Duration of post-treatment incubation (if applicable):
- Subsequently the skin tissues were incubated for 42 hours at 37°C.
- Number of replicates:
- Triplicate; treatment and concurrent negative control and positive control groups
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- mean (n=3)
- Value:
- 82
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Basis: mean. Time point: 15 minute exposure. Remarks: n=3; SD = 1.3% ; Score in terms of percentage of negative control.
- Other effects / acceptance of results:
- - OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Screened prior to test. Not applicable.
DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory previously demonstrated technical proficiency of the validated method with proficiency test items (information in the public domain). Additionally, concurrent positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data are presented in the full study report. All values for the control groups were within the ranges of the current Historic Control Data. Historic Control Data (n=138): the mean OD of the positive control was 0.108 ; range 0.023 to 0.449. In this same period the mean OD of the negative control was 0.986 ; range 0.422 – 1.547. - Interpretation of results:
- GHS criteria not met
- Remarks:
- Criteria used for interpretation of results: EU
- Conclusions:
- Under the conditions of this study, the test item is not considered to be irritating to the skin.
- Executive summary:
The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of 3 tissues per test item together with negative and positive controls. 25 μL of the undiluted test item was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control), respectively. The positive control was re-spread after 7 minutes contact time. Use of 25 µL test item, negative control and positive controls (ca. 65 µL/cm2), respectively rather than 10 µL (ca. 26 µL/cm2) protocol deviation was not considered to affect the reliability of the study. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability of 8.4% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 82%. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 16.0%, indicating that the test system functioned properly. Since the mean relative tissue viability for the test item was above 50% after 15 minutes treatment it is not considered to be irritant. Under the conditions of this study the test item is not irritant to the skin.
Reference
Table 1. Mean absorption and tissue viability in the in vitro skin irritation test with the test item
Item |
OD570 of tissues |
Mean OD570 of triplicate tissues |
± SD of OD570 |
Relative mean viability (%) |
± SD of Relative mean viability (%) |
Negative Control Item |
1.2232 |
1.0336 |
0.165 |
100* |
16 |
0.9583 |
|||||
0.9193 |
|||||
Positive Control Item |
0.1091 |
0.0868 |
0.013 |
8.4 |
1.9 |
0.0803 |
|||||
0.0711 |
|||||
Test Item |
0.8577 |
0.8472 |
0.020 |
82 |
1.3 |
0.8324 |
|||||
0.8516 |
OD = Optical Density
SD = Standard deviation
* = The mean viability of the negative control tissues is set at 100%.
Values are corrected for background absorption (0.043). Isopropanol was used to measure background adsorption.
Negative control: Phosphate buffered saline (PBS)
Positive control: 5% (aq.) Sodium dodecyl sulphate (SDS)
The relative mean tissue viability for the positive control treated tissues was 8.4% relative to the negative control following the 15-minute exposure/42-hour incubation period. With standard deviation of 1.9%. The positive control acceptance criterion was therefore satisfied. The negative control triplicate treated tissues mean OD570 was 1.0336 and the standard deviation of viability was 16%. The acceptance criterion was therefore satisfied. The test item triplicate treated tissues viability standard deviation was 1.3%. The mean OD570 for both the negative and positive controls were within the historical control ranges. All assay acceptance criteria were met.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 20-02-2020 to 17-04-2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Guideline study performed under GLP. All relevant validity criteria were met.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: May 2018 ; signature: August 2018
- Species:
- chicken
- Strain:
- not specified
- Details on test animals or tissues and environmental conditions:
- SOURCE OF COLLECTED EYES
- Source: Recognised supplier (documented in the full study report)
- Number of animals: Not reported.
- Characteristics of donor animals (e.g. age, sex, weight): ca. 7 weeks old (typically).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Freshly isolated heads from ca. 7 week old donors were collected from the abattoir at transported at ambient. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at the test facility and processed within 2 hours of collection by the test facility. After removing the individual head from the plastic box, it was put on soft paper. The eyelids were carefully removed, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit (by standardised procedure), then the eye was placed onto damp paper and the nictitating membrane was removed with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained. Eyes where then clamped in the superfusion apparatus and then re-examined to ensure good condition. Then the eyes were acclimatised for 45 to 60 minutes at (32±1.5°C)
- Time interval prior to initiating testing: < 24 hours. Eyes were prepared for testing immediately on same day arrival within 2 hours of collection.
- indication of any existing defects or lesions in ocular tissue samples: None. Only eyes free from damage
- Indication of any antibiotics used: None reported. - 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): 0.03 mL (or 30 µL of the test item)
- Concentration (if solution): undiluted
VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable. - Duration of treatment / exposure:
- The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of physiological saline.
Controls (negative and positive control items) were similarly applied to the cornea in the negative and positive control groups respectively. - Observation period (in vivo):
- Treated eyes were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been rinsed with the physiological saline.
- Number of animals or in vitro replicates:
- Triplicate (n=3)
- Details on study design:
- SELECTION AND PREPARATION OF ISOLATED EYES: Full details are provided in the full study report. Freshly isolated heads from ca. 7 week old donors were collected from the abattoir at transported at ambient. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at the test facility and processed within 2 hours of collection by the test facility. After removing the individual head from the plastic box, it was put on soft paper. The eyelids were carefully removed, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit (by standardised procedure), then the eye was placed onto damp paper and the nictitating membrane was removed with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained. Eyes where then clamped in the superfusion apparatus and then re-examined to ensure good condition. Then the eyes were acclimatised for 45 to 60 minutes at (32±1.5°C)
EQUILIBRATION AND BASELINE RECORDINGS: Taken at zero time after 45 to 60 minutes incubation prior to exposure.
NUMBER OF REPLICATES: Triplicate (3) for test item, positive control and negative controls.
NEGATIVE CONTROL USED: Yes.
SOLVENT CONTROL USED (if applicable): Not applicable.
POSITIVE CONTROL USED : Benzalkonium chloride (5%)
APPLICATION DOSE AND EXPOSURE TIME: Application dose: 0.03 mL (or 30 µL of the test item) ; Exposure time: 10 seconds.
OBSERVATION PERIOD: prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after decontaminated with saline.
REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Test item remained in place for 10 seconds and then rinsed with 20 mL physiological saline. Treated eyes were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes after the eyes had been rinsed with the physiological saline.
- Indicate any deviation from test procedure in the Guideline: Not applicable.
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Microscope.
- Damage to epithelium based on fluorescein retention: Microscope.
- Swelling: measured with optical pachymeter on a slit-lamp microscope
- Macroscopic morphological damage to the surface: Yes.
- Others (e.g, histopathology): Not applicable.
SCORING SYSTEM:
- Mean corneal swelling (%): See tables 1 to 2.
- Mean maximum opacity score: See tables 1 to 2.
- Mean fluorescein retention score at 30 minutes post-treatment: See tables 1 to 2.
DECISION CRITERIA: In accordance with guideline OECD TG 438.
Endpoints used during the evaluation procedure were corneal opacity, swelling, fluorescein retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium, sticking of test item to the cornea). All of the endpoints, with the exception of fluorescein retention (which is only determined at 30 minutes after test item exposure) were determined at each of the above time points.
ICE classes were determined based on a predetermined range in accordance with the criteria given in OECD TG 438.
The overall in vitro irritancy classification of the test item was determined by using all the classification information and criteria given in OECD TG 438 and the UN GHS classification referenced in the guideline. Full details are provided in the full study report.
A test was considered acceptable if the concurrent negative or vehicle/solvent items and the concurrent positive controls were within the historic control data range and/or identified as GHS Non Classified and GHS Category 1, respectively - Irritation parameter:
- cornea opacity score
- Run / experiment:
- mean (n=3)
- Value:
- 0
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: ICE Class I
- Irritation parameter:
- fluorescein retention score
- Run / experiment:
- mean (n=3)
- Value:
- 0
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: ICE Class I
- Irritation parameter:
- percent corneal swelling
- Run / experiment:
- mean (n=3)
- Value:
- -3.3
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: value %; ICE Class I
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None reported.
DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory previously demonstrated technical proficiency of the validated method with proficiency test items (information in the public domain). Additionally, concurrent positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline: Not applicable. Applicant assessment of the data indicates that the positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively. - Interpretation of results:
- GHS criteria not met
- Remarks:
- Criteria used for interpretation of results: EU
- Conclusions:
- Under the conditions of this study, the test item was considered to be non-irritant.
- Executive summary:
The study was performed according to OECD TG 438 in accordance with GLP to assess the eye irritancy potential of the test item in isolated chicken eyes. The method involves evaluation of the eye hazard potential of a test chemical as measured by its ability to induce toxicity in an enucleated chicken eye. Toxic effects to the cornea are measured by (i) a qualitative assessment of opacity (ii) a qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention) (iii) a quantitative measurement of increased thickness (swelling) and (iv) a qualitative evaluation of macroscopic morphological damage to the surface. The method is also able to identify substances not requiring UN GHS classification. After the zero reference measurements, the eyes were held in a horizontal position and the test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds exposure time, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 μL test item. The three positive control eyes were treated in a similar way with 30 μL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated. For the test item the mean corneal opacity was 0.0 (ICE Class I). The mean fluorescein retention was 0.0 (ICE Class I) and the mean corneal thickness across 30, 75, 120, 180 and 240 minutes was ICE Class V with maximal corneal swelling -3.3%. No other morphological effect was observed. The negative control gave a prediction of GHS non-classified for eye irritation (ICE Class I) across all categories. The positive control gave a prediction of GHS Category 1 (ICE Class IV across cornel opacity and fluorescein retention categories and Class III in mean corneal thickness) signifying that the test system performed adequately. Under the conditions of this study, the test item was considered to be non-irritant.
Reference
Table 1. Ocular reactions
Test Item |
|
|
Maximal mean score for corneal opacity |
0.00 |
ICE Class I |
Mean score of Fluorescein Retention |
0.00 |
ICE Class I |
Corneal swelling |
-3.3% |
ICE Class I |
|
|
|
Positive Control Item |
|
|
Maximal mean score for corneal opacity |
3.83 |
ICE Class IV |
Mean score of Fluorescein Retention |
2.83 |
ICE Class IV |
Corneal swelling |
22.9% |
ICE Class III |
|
|
|
Negative Control Item |
|
|
Maximal mean score for corneal opacity |
0.00 |
ICE Class I |
Mean score of Fluorescein Retention |
0.00 |
ICE Class I |
Corneal swelling |
0.00% |
ICE Class I |
- Corneal Opacity Scores
Maximum score was 0.0 (n=3 of 3) in the test item group. Maximum score was 0.0 in the negative control. In the positive control group the Maximum score was 4.0. No morphological effects were noted in the test item or negative control item treated eyes. In the positive control group, no morphological effects were noted.
- Fluorescein Retention Scores
Maximum score was 0.5 (n=1 of 3) in the test item group and the negative control. In the positive control group the Maximum score was 3.00.
Table 2. Individual scoring - test item
End Point |
Eye Number |
Time (after decontamination) |
|||||
0 minutes |
30 minutes |
75 minutes |
120 minutes |
180 minutes |
240 minutes |
||
Corneal Opacity |
12 |
0 |
0 |
0 |
0 |
0 |
0 |
13 |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
|
14 |
0 |
0 |
0 |
0.0 |
0.0 |
0.0 |
|
Mean difference (time - 0 min) |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
ICE Class |
I |
||||||
Fluorescein Retention |
12 |
0.0 |
0.0 |
|
|
|
|
13 |
0.5 |
0.5 |
|
|
|
|
|
14 |
0.0 |
0.0 |
|
|
|
|
|
Mean difference (30 - 0 min) |
|
0.00 |
|
|
|
|
|
ICE Class |
I |
||||||
Corneal Thickness |
12 |
0.0% |
1.6% |
0.0% |
-1.6% |
-3.3% |
-3.3% |
13 |
0.0% |
0.0% |
-3.3% |
-3.3% |
-3.3% |
-3.3% |
|
14 |
0.0% |
0.0% |
-1.7% |
-1.7% |
-3.3% |
-3.3% |
|
Mean |
0.0% |
0.5% |
-1.7% |
-2.2% |
-3.3% |
-3.3% |
|
Mean Corneal Swelling (%) |
|
0.5 |
-1.7 |
-2.2 |
-3.3 |
-3.3 |
|
ICE Class |
I |
||||||
ICE Classes Combined: |
3 x I |
||||||
Classification: |
Category 1 |
The test was considered acceptable since the concurrent negative or vehicle/solvent items and the concurrent positive controls were identified as GHS Non Classified and GHS Category 1, respectively.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Skin Irritation :
Key study : in vitro, OECD TG 439, 2020 : The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of 3 tissues per test item together with negative and positive controls. 25 μL of the undiluted test item was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control), respectively. The positive control was re-spread after 7 minutes contact time. Use of 25 µL test item, negative control and positive controls (ca. 65 µL/cm2), respectively rather than 10 µL (ca. 26 µL/cm2) protocol deviation was not considered to affect the reliability of the study. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability of 8.4% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 82%. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 16.0%, indicating that the test system functioned properly. Since the mean relative tissue viability for the test item was above 50% after 15 minutes treatment it is not considered to be irritant. Under the conditions of this study the test item is not irritant to the skin.
Eye irritation/corrosion :
Key study : in vitro, OECD TG 438, 2020 : The study was performed according to OECD TG 438 in accordance with GLP to assess the eye irritancy potential of the test item in isolated chicken eyes. The method involves evaluation of the eye hazard potential of a test chemical as measured by its ability to induce toxicity in an enucleated chicken eye. Toxic effects to the cornea are measured by (i) a qualitative assessment of opacity (ii) a qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention) (iii) a quantitative measurement of increased thickness (swelling) and (iv) a qualitative evaluation of macroscopic morphological damage to the surface. The method is also able to identify substances not requiring UN GHS classification. After the zero reference measurements, the eyes were held in a horizontal position and the test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds exposure time, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 μL test item. The three positive control eyes were treated in a similar way with 30 μL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated. For the test item the mean corneal opacity was 0.0 (ICE Class I). The mean fluorescein retention was 0.0 (ICE Class I) and the mean corneal thickness across 30, 75, 120, 180 and 240 minutes was ICE Class V with maximal corneal swelling -3.3%. No other morphological effect was observed. The negative control gave a prediction of GHS non-classified for eye irritation (ICE Class I) across all categories. The positive control gave a prediction of GHS Category 1 (ICE Class IV across cornel opacity and fluorescein retention categories and Class III in mean corneal thickness) signifying that the test system performed adequately. Under the conditions of this study, the test item was considered to be non-irritant.
Respiratory Irritation:
No data available.
Justification for classification or non-classification
The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for skin irritation
The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for eye irritation
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.