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EC number: 212-295-5 | CAS number: 778-28-9
- 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:
- 30 July 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guideline
- 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)
- Version / remarks:
- 9 October 2017
- Deviations:
- no
- GLP compliance:
- yes
Test material
- Reference substance name:
- Butyl toluene-4-sulphonate
- EC Number:
- 212-295-5
- EC Name:
- Butyl toluene-4-sulphonate
- Cas Number:
- 778-28-9
- Molecular formula:
- C11H16O3S
- IUPAC Name:
- butyl 4-methylbenzene-1-sulfonate
- Test material form:
- liquid
- Remarks:
- (colourless to slightly yellow)
- Details on test material:
- - Storage Conditions: room temperature protected from light
Constituent 1
Test animals / tissue source
- Species:
- cattle
- Strain:
- not specified
- Details on test animals or tissues and environmental conditions:
- SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from a slaughterhouse (Vitelco, 's-Hertogenbosch, The Netherlands). The eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
Test system
- 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): 750 µL
NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL
POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL - Duration of treatment / exposure:
- 10 ± 1 minutes
- Duration of post- treatment incubation (in vitro):
- 120 ± 10 minutes
- Number of animals or in vitro replicates:
- Three corneas were allocated to the test material, three corneas were allocated to the negative control item, and three corneas were allocated to the positive control item.
- Details on study design:
- PREPARATION AND SELECTION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium containing 1 % (v/v) L-glutamine and 1 % (v/v) Foetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1 °C. The corneas were incubated for a minimum of 1 hour at 32 ± 1 °C.
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.
NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.
TREATMENT METHOD
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control or test material was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or test material over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1 °C.
REMOVAL OF TEST SUBSTANCE
After the incubation the solutions were removed and the epithelium was washed with MEM (Earle’s Minimum Essential Medium) with phenol red and thereafter with cMEM. Possible pH effects of the test material on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM.
POST-EXPOSURE INCUBATION
Corneas were incubated for 120 ± 10 minutes at 32 ± 1 °C.
METHODS FOR MEASURED ENDPOINTS
After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.
- Opacity measurement: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device OP-KIT) was calculated according to the following equation:
Opacity = [(I0/I) - 0.9894] / 0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test material or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test material or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Application of Sodium Fluorescein: Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1 °C.
- Permeability Determinations: After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test material was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.
INTERPRETATION
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine whether the test material induced irritation through only one of the two endpoints.
The test material was classified according to the following prediction model:
IVIS ≤ 3: No Category (UN GHS)
IVIS >3; ≤ 55: No prediction can be made (UN GHS)
IVIS > 55: Category 1 (UN GHS)
ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
Results and discussion
In vitro
Resultsopen allclose all
- Irritation parameter:
- in vitro irritation score
- Run / experiment:
- Test material (mean)
- Value:
- 0.4
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation parameter:
- in vitro irritation score
- Run / experiment:
- Negative control (mean)
- Value:
- 1.6
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation parameter:
- in vitro irritation score
- Run / experiment:
- Positive control (mean)
- Value:
- 55
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Other effects / acceptance of results:
- The corneas treated with the test material showed opacity values ranging from -0.2 to 0.5 and permeability values ranging from 0.006 to 0.018. The corneas were clear after the 10 minutes of treatment with the test material. No pH effect of the test material was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -0.1 to 0.7 after 10 minutes of treatment with the test material. The test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.4 after 10 minutes of treatment.
The individual in vitro irritancy scores for the negative controls ranged from 0.5 to 2.6. One of the negative control eyes was excluded from the analysis since the opacity was above the historical data base which resulted in an IVIS outside the historical data base. The corneas treated with the negative control item were clear after the 10 minutes of treatment.
The individual positive control in vitro irritancy scores ranged from 54 to 61. The corneas treated with the positive control item were turbid after the 10 minutes of treatment.
ACCEPTANCE OF RESULTS:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol) was 55 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
Any other information on results incl. tables
Summary of results
Treatment |
Final opacity |
Final OD490 |
In vitro Irritancy Score |
Negative control |
2.7 |
-0.005 |
2.6 |
0.5 |
-0.003 |
0.5 |
|
5.2* |
0.029* |
5.7* |
|
Mean |
1.6 |
-0.004 |
1.6 |
Positive control |
21 |
2.218 |
54 |
22 |
2.592 |
61 |
|
30 |
1.370 |
50 |
|
Mean |
24 |
2.060 |
55 |
Test material |
0.5 |
0.013 |
0.7 |
-0.2 |
0.006 |
-0.1 |
|
0.2 |
0.018 |
0.5 |
|
Mean |
0.2 |
0.012 |
0.4 |
Positive control and test material are corrected for the negative control.
*Excluded from analysis
Applicant's summary and conclusion
- Interpretation of results:
- other: not classified according to EU criteria
- Conclusions:
- Under the conditions of the study, the test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.4 after 10 minutes of treatment.
In conclusion, since the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage. - Executive summary:
The eye irritation potential of the test material was evaluated, as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). The study was conducted in accordance with the standardised guideline OECD 437 and under GLP conditions.
During the study, the test material was applied as supplied (750 μL) directly on top of the corneas. The eye damage of the test material was tested through topical application for 10 minutes. After exposure the cornea was thoroughly rinsed to remove the test material and incubated for 2 hours with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol) was 55 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
Under the conditions of the study, the test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.4 after 10 minutes of treatment.
In conclusion, since the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
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