Registration Dossier

Administrative data

Description of key information

The skin irritation profile of benzene, tetrapropylene-, distn. residues, sulfonated, sodium salts (registered/target substance) was determined in an in vitro corrosivity (OECD 435) and skin irritation (OECD 439) assays. An in vivo rabbit study (OECD 404) on a read across source substance confirmed these skin irritation results. The eye irritation profile of the registered/target substance was determined by eye in vitro toxicity studies following OECD guidelines 437 (Bovine Corneal Opacity and Permeability Test) and 492 (Reconstructed Human Cornea-like Epithelium Test). Several in vivo rabbit studies on read across source substance confirmed these eye non-irritation results.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Oct 2017 - 20 Oct 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study completed according to OECD 435 Guidelines, and under GLP.
Qualifier:
according to
Guideline:
OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)
GLP compliance:
yes (incl. certificate)
Test system:
other: Corrositex Kit Biobarriers
Source species:
other:
Cell type:
other:
Cell source:
other:
Vehicle:
water
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Species:
other: N/A - Macromolecular Barrier
Type of coverage:
other: In vitro
Vehicle:
water
Remarks:
Tissue culture water
Controls:
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
500 μl of the test article to each of four test vials containing biobarriers
Duration of treatment / exposure:
Single Exposure
Observation period:
The amount of time required for the test article to penetrate or destroy each biobarrier was recorded and the mean time of the four replicates is used to designate the United Nations (U.N.) Packing Group classification and GHS Sub-category
Details on study design:
Background
The Corrositex® test is a standardized and reproducible method that can be employed to determine the potential corrosivity and the Packing Group classification of specified categories of chemical compounds under the hazardous materials transportation regulations administered by the U.S. Department of Transportation (DOT) and international dangerous goods codes. The Corrositex® test predicts the in vivo corrosive potential of a chemical compound or mixture by using as an endpoint the amount of time it takes for a chemical to destroy a synthetic biobarrier. A color change in a proprietary liquid Chemical Detection System (CDS) is used to indicate that the chemical has passed through the biobarrier.

Procedure
The Corrositex® test is a three-step procedure. First, the test article was qualified to ensure that it was compatible with the Corrositex® system, and then it was categorized according to pH to determine cut-off times. Finally, it was classified based on the mean time the test article took to penetrate the biobarriers.

Qualification
150 μl of the test article were added to the CDS reagent in a Qualify vial, and the vial was observed for any notable color change. An observable color change indicates that the test article is compatible with the Corrositex® system.

Categorization
Next, the test article was categorized, which determined cut-off times for the Packing Group designations. A 10% formulation of the test article was prepared and its pH was measured. If the pH of the 10% formulation was <7.0, 150 μl of the neat test article were added to Vial A. The pH of Vial A was then measured, and if it was lower or equal to 5.0, the test article was assigned to Category 1, and if it was >5.0, the test article was assigned to Category 2. If the pH of the 10% formulation was >7.0, 150 μl of the neat test article were added to Vial B. The pH of Vial B was measured, and if the final pH was 9.0, the test article was Category 1, and if it was greater or equal than 9.0, the test article was Category 2.

Classification
Finally, the test article was classified to determine the Packing Group by adding 500 μl of the test article to each of four test vials containing biobarriers. If the test article destroys the biobarrier, it will come in contact with the CDS reagent in the vial and induce a color change similar to that observed in the Qualify vial. The amount of time required for the test article to penetrate or destroy each biobarrier was recorded and the mean time of the four replicates is used to designate the United Nations (U.N.) Packing Group classification and GHS Sub-category as described below. A positive control was performed using 1.0 N sodium hydroxide. The result for the positive control is considered valid if it falls within the range of the MB Research historical mean ± 2 standard deviations. A negative control was performed using 1% citric acid; the result is considered valid if the breakthrough time is greater than 60 minutes.
Irritation / corrosion parameter:
penetration time (in minutes)
Run / experiment:
1
Value:
> 85.21
Vehicle controls validity:
valid
Negative controls validity:
valid
Remarks:
Citric acid
Positive controls validity:
valid
Remarks:
1.0 N Sodium hydroxide
Remarks on result:
no indication of irritation
Remarks:
Non Corrosive

The 1.0 N sodium hydroxide" positive control had a breakthrough time of 16.15 minutes, which fell within the range allowed (>13.7 and <22.2 minutes). The 1% citric acid negative control had a breakthrough time of 68 minutes, which met the acceptance criterion of >60 minutes.

Interpretation of results:
GHS criteria not met
Remarks:
The material was considered non-corrosive.
Conclusions:
The material was considered non-corrosive.
Executive summary:

Benzene, tetrapropylene-, distn. residues, sulfonated, sodium salt was analyzed using the Corrositex® test method to determine its dermal corrosivity potential and U.N. Packing Group classification and GHS Sub-category. The 1.0 N sodium hydroxide" positive control had a breakthrough time of 16.15 minutes, which fell within the range allowed (>13.7 and <22.2 minutes). The 1% citric acid negative control had a breakthrough time of 68 minutes, which met the acceptance criterion of >60 minutes. The results of this study indicated that the test article was compatible with the Corrositex® system. Based on the pH of the test article, it was assigned as a Corrositex® Category 2 test article. The results obtained from the evaluation of four replicate tests demonstrated that a mean time of >85.21 minutes was required to penetrate the synthetic biobarriers. These findings lead to the classification of this test article as a Non-Corrosive material.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Noc 2017 - 01 Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study completed according to OECD 439 Guidelines, and under GLP.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
OECD Guideline 439 (In Vitro Skin Irritation)
Deviations:
no
Principles of method if other than guideline:
OECD Guideline 439 (In Vitro Skin Irritation)
GLP compliance:
yes (incl. certificate)
Species:
human
Vehicle:
other: vehicle
Controls:
yes, concurrent vehicle
Amount / concentration applied:
30 μl of the test article were applied to the EpiDerm™ tissue
Duration of treatment / exposure:
MatTek EpiDerm™ tissue samples were treated in triplicate with the test article, negative control and
positive control for 60 minutes
Observation period:
EpiDerm™ tissues were returned to the incubator for 24±2 hours. Medium was changed at 24±2 hours. Tissues were returned to the incubator for an additional 18±2 hours
Details on study design:
EpiDerm™ Tissue Samples
EpiDerm™ tissues, Lot No. 27631, Kit F, were received from MatTek on 28 Nov 2017 and refrigerated
at 2-8°C. Before use, the tissues were incubated (37±1°C, 5±1% CO2) with assay medium (MatTek) for a one-hour equilibration. The tissues were then moved to new wells with fresh medium for an additional overnight equilibrium, for 18±3 hours. Equilibration medium was replaced with fresh medium before dosing.

Test Article Reduction of MTT
100 μl of the test article were mixed with 1 ml of MTT solution (1 mg/ml methyl thiazole tetrazolium diluted in Dulbecco's Modified Eagle's Medium [DMEM]). A negative control (100 μl tissue culture water) was tested concurrently. The solution was incubated at room temperature in the dark for 60 minutes. After incubation, the solution was visually inspected for purple coloration, which indicates that the test article reduced MTT. Since tissue viability is based on MTT reduction, direct reduction
by a test article can exaggerate viability, making a test article seem less irritating than its actual irritation potential. The test article did not reduce MTT and the assay continued as per the protocol.

Mesh Compatibility
Pre-cut nylon mesh supplied with the tissues was placed on a slide and 30 μl of the test article or tissue culture water (negative control) were applied. After 60 minutes of exposure, the mesh was checked microscopically. No interaction between the test article or phosphate-buffered saline and the mesh was observed so the test article was dosed using the mesh as a spreading aid.

Dosing
30 μl of the test article were applied to the EpiDerm™ tissue. A nylon mesh was then placed on top to facilitate even distribution of the test article. A negative control (30 μl of PBS) and a positive contr ol (30 μl of 5% SDS solution) were each tested concurrently, with a nylon mesh placed on top to facilitate even distribution of the material. Each treatment with test article or control was conducted in triplicate. The exposure period for the test article and controls was 60 minutes. The dosed tissues were placed in an incubator at 37±1°C, 5±1% CO2 for 35±1 minute, and then returned to the sterile hood for the remainder of the 60-minute exposure period. After dosing and incubation, the tissues were rinsed with PBS, blotted to remove the test substance and dry the tissue, and transferred to fresh medium. The rinsed EpiDerm™ tissues were returned to the incubator for 24±2 hours. Medium was
changed at 24±2 hours. Tissues were returned to the incubator for an additional 18±2 hours.

Tissue Viability (MTT Reduction)
At the end of the incubation period, each EpiDerm™ tissue was rinsed with PBS and transferred to a 24-well plate containing 300 μl of MTT solution (1 mg/ml MTT in DMEM). The tissues were then re turned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period , each EpiDerm™ tissue was rinsed with PBS and then treated with 2.0 ml of extractant solution (isopropanol) per well for at least two hours, with shaking, at room temperature. Two aliquots of the extracted MTT formazan were measured at 540 nm using a plate reader (μQuant Plate Reader, BioTek Instruments, Winooski, VT).

Analysis of Data
The mean absorbance value for each time point was calculated from the optical density (OD) of the duplicate samples and expressed as percent viability for each sample using the following formula: %
viability = 100 X (OD sample/OD negative control)

Quality Controls
The assay meets the acceptance criteria if the mean OD540 of the negative control tissues is be tween 1.0 and 2.5, inclusive, and the mean viability of positive control tissues, expressed as perc entage of the negative control tissues, is at least 20%. In addition, the standard deviation (SD) calculated from individual percent tissue viabilities of the three identically-treated replicates must be less than 18%.

Skin Irritation Prediction
According to the EU and GHS3 classification (R38 / Category 2 or no label), an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is 50% or less of the mean viability of the negative controls.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
ca. 95.7
Negative controls validity:
valid
Remarks:
97.2% - PBS
Positive controls validity:
valid
Remarks:
3.3% - SDS
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
ca. 15.9
Negative controls validity:
valid
Remarks:
102.5% - PBS
Positive controls validity:
valid
Remarks:
3.5% - SDS
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
ca. 13.9
Negative controls validity:
valid
Remarks:
100.3% - PBS
Positive controls validity:
valid
Remarks:
3.2% - SDS
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The mean OD540 of the negative control tissues was 2.247, and the mean viability of positive control tissues was 3.3%. The standard deviation (SD) calculated from individual percent tissue viabilities of the three identically-treated replicates was 2.69% for the negative control and 0.15% for the positive control. All controls passed the acceptance criteria.

Of the three test-article treated tissues, the individual viabilities were 95.1%, 15.9% and 13.9%, with a mean tissue viability of 41.6%. The protocol states that the standard deviation of the three treated tissues be <18%. The standard deviation of the three treated tissues was 46.3%. Although the standard deviation did not meet the acceptance criterion, 2 of the 3 tissues had similar viability and the mean was 41.6%. Therefore, the material was classified as an Irritant
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Of the three test-article treated tissues, the individual viabilities were 95.1%, 15.9% and 13.9%, with a mean tissue viability of 41.6%. The protocol states that the standard deviation of the three treated tissues be <18%. The standard deviation of the three treated tissues was 46.3%. Although the standard deviation did not meet the acceptance criterion, 2 of the 3 tissues had similar viability and the mean was 41.6%. Therefore, the material was classified as an Irritant.
Executive summary:

To predict dermal irritation potential, MatTek EpiDerm tissue samples were treated with 30 μl of benzene, tetrapropylene-, distn. residues, sulfonated, sodium salts, 30 μl of phosphate buffered saline [PBS] (negative control), or 5% sodium dodecyl sulfate [SDS] solution (positive control). The samples were then incubated in 300 μl Methyl thiazole tetrazolium (MTT) solution (1 mg/ml MTT in DMEM) followed by 2.0 mL extractant solution (isopropanol). Mean tissue viability was determined to be 41.6% for the test material, 3.3% for the positive control, and 100% for the negative control. The material was concluded to be a skin irritant.

Endpoint:
skin irritation: in vivo
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No report on this study was provided to WRc, therefore this data has not been reviewed. All data provided in this study record was entered by the data owner.
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH 1. HYPOTHESIS FOR THE CATEGORY APPROACH The read across follows Scenario 5 - Qualitatively and quantitatively similar effects are caused by a common compound, which is formed from all category members (as described in the 2017 Read-Across Assessment Framework document). 2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES) TARGET: Benzene, tetrapropylene-, distn. residues, sulfonated, sodium salts SOURCE: Benzenesulfonic acid, methyl-, mono-C20-24-branched alkyl derivs., calcium salts. 3. CATEGORY APPROACH JUSTIFICATION Linear and non-linear or branched alkylbenzene sulfonates are anionic surfactants with molecules characterized by a hydrophobic (apolar) and a hydrophilic (polar) group. As a group of chemicals, they are generally mixtures of closely related isomers and homologues. Each molecule contains an aromatic ring sulfonated at the para position and attached to either a linear or a branched alkyl chain at any position except the terminal carbons. The sulfonate group is a common functional group present in each of the category members, and is expected to exhibit similar biological activities with little influence from the length of carbon chain. The cation components of the chemicals (e.g. calcium, magnesium, sodium, or barium) are not expected to contribute significantly to the toxicity. 4. DATA MATRIX See Read Across document attached to CSR
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
See study report attached.
Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
Undiluted material - 0.5 mL
Duration of treatment / exposure:
4 hours
Observation period:
72 hours
Number of animals:
6 animals
Details on study design:
See study report attached.
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
ca. 2.6
Remarks on result:
probability of moderate irritation
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
ca. 3.3
Remarks on result:
probability of moderate irritation
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
24/48/72 h
Score:
ca. 6.1
Remarks on result:
probability of moderate irritation
Irritant / corrosive response data:
Moderate-severe to severe erythema and moderate-severe edema reactions. Subcutaneous hemorrhaging, blanching, desquamationa, and possible necrotic areas were also observed. Slight dermal irritation continue to be present at three sites at the Day 14 observation.
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Skin Irritant (GHS - Category 2, EU - R38)
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 October 2017 - 19 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes (incl. certificate)
Species:
cattle
Strain:
not specified
Remarks:
bovine cornea
Details on test animals or tissues and environmental conditions:
Test System
The bovine eyes, in a Hanks’ Balance Salt Solution (HBSS) with penicillin-streptomycin, were received and transported to MB Research in a refrigerated container.

Pretest Procedures
Fresh assay solutions were prepared prior to use. MEM solution was prepared by stirring together one jar of MEM powder (sufficient to make one liter of solution), 2.2 g of sodium bicarbonate, 0.292 g L-glutamine, 10 ml of fetal bovine serum (FBS) and brought to a final volume of 1000 ml with distilled water. The MEM solution was kept in a 32 (±1)°C incubator for the duration of testing. HBSS was prepared by stirring together HBSS powder (sufficient to make one liter) and 0.35 g of sodium bicarbonate and brought to a final volume of 1000 ml with distilled water. HBSS was maintained at room temperature. In addition, MEM solution with phenol red was prepared by stirring together 9.3 g of MEM with phenol red (sufficient to make one liter), 2.2 g of sodium bicarbonate, 0.292 g L-glutamine, 10 ml of fetal bovine serum (FBS) and brought to a final volume of 1000 ml with distilled water. The MEM solution with phenol red was kept in a 32 (±1)°C incubator for the duration of testing. The eyes were examined prior to use on the day of dosing. Any eye with a cornea exhibiting evidence of vascularization, pigmentation, opacity or scratches was discarded. Corneas from eyes that were free of defects were dissected from the surrounding tissues. A 2-3 mm rim
of sclera was left attached to each cornea. The corneas were then placed in a container of fresh HBSS. The dissected corneas were mounted in specially designed holders that were separated into anterior and posterior chambers and filled separately. Each cornea was mounted allowing the epithelium of the cornea to project into the anterior chamber. The posterior chamber was filled with MEM solution ensuring contact with the endothelium. The anterior chamber was filled with MEM solution, ensuring contact with the epithelium. Each cornea was visually inspected again to ensure there were no defects. The entire holder was incubated at 32 (±1)°C and allowed to equilibrate for at least one hour, but not longer than two hours. A pre-exposure determination of opacity was made for each cornea by measuring each against the blank supplied by the opacitometer. Any cornea with a value greater than 7 units was discarded.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 ml of test article, Minimal Essential Media (MEM, negative control), or 100% ethanol (positive control)
Duration of treatment / exposure:
Following a 10-minute exposure for each group of dosed corneas, opacity measurements and sodium fluorescein permeability were determined.
Duration of post- treatment incubation (in vitro):
All corneas were incubated at 32 (±1)°C for an additional two hours at which time the MEM solution in the anterior and posterior chambers was removed and the holders refilled with fresh MEM solution. After 10 (±1) minutes), the test article, ethanol or MEM was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were then refilled with fresh MEM solution. Opacity measurements were made following the 10-minute exposure and MEM solution refill.
Number of animals or in vitro replicates:
Three bovine corneas per group
Details on study design:
Study Procedure
Following the pretest observations, the MEM solution was removed from the anterior chamber. A volume of 0.75 ml of test article, ethanol, or MEM solution was applied to the epithelium of each of the three test article corneas, three positive control corneas and three negative control corneas in a manner which ensured that the entire cornea was covered. Test article corneas were dosed via the open-chamber method. The negative and positive controls were dosed via the closed-chamber method. All holders and corneas were placed in a horizontal position (anterior side up) in the 32 (±1)°C incubator. After 10 (±1) minutes), the test article, ethanol or MEM was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were then refilled with fresh MEM solution. Opacity measurements were made following the 10-minute exposure and MEM solution refill.

All corneas were incubated at 32 (±1)°C for an additional two hours at which time the MEM solution in the anterior and posterior chambers was removed and the holders refilled with fresh MEM solution. A measurement of opacity was taken with each treated cornea compared to the blank supplied with the OP-KIT. This reading was used in the final IVIS calculations. Immediately following the two-hour opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 ml of 0.4% sodium fluorescein solution in Dulbecco's phosphatebuffered saline (PBS). Each holder was then returned to the 32 (±1)°C incubator in a horizontal position (anterior side up) ensuring contact of the fluorescein with the cornea. After 90 (±5) minutes, the fluid from the posterior chamber was removed and the amount of dye that passed through the cornea (permeability) was measured as the optical density at 490 nm by a spectrophotometer. A 1:1000 dilution of the fluorescein was prepared and measured in the spectrophotometer as a measure of consistency.
Irritation parameter:
cornea opacity score
Remarks:
Permeability
Run / experiment:
1
Value:
25.333
Negative controls validity:
valid
Remarks:
Minimal Essential Media; Corneal opacity score=0.00
Positive controls validity:
valid
Remarks:
100% Ethanol, Corneal opacity score 13.33
Remarks on result:
no indication of irritation
Remarks:
Non-Corrosive
Irritation parameter:
in vitro irritation score
Remarks:
IVIS
Run / experiment:
2
Value:
26.04
Negative controls validity:
valid
Remarks:
Value= 0.35
Positive controls validity:
valid
Remarks:
Value= 25.83
Remarks on result:
no indication of irritation
Remarks:
Non-Corrosive

The ethanol positive control IVIS was 25.83, which fell within the acceptance range of 16.96 - 37.00 (± 2 standard deviations of the historical mean).

Interpretation of results:
GHS criteria not met
Remarks:
The test material is not considered an ocular corrosive or severe irritant (Category 1).
Conclusions:
The test material is not considered an ocular corrosive or severe irritant (Category 1).
Executive summary:

To determine the potential for ocular irritation using an alternative to the Draize methodology, a study based on the methodology described in the current OECD Guideline for the Testing of Chemicals No. 437 was conducted. Three bovine corneas per group were dosed with 0.75 ml of benzene, tetrapropylene-, distn. residues, sulfonated, sodium salts, Minimal Essential Media (MEM, negative control), or 100% ethanol (positive control). Following a 10-minute exposure for each group of dosed corneas, opacity measurements and sodium fluorescein permeability were determined. The MEM solution is then removed from the anterior chamber and replaced with 1.0 ml of 0.4% sodium fluorescein solution. After 90 min the fluid from the posterior chamber is removed and the amount of dye that passed through the cornea is measured as the optical density at 490 nm by a plate reader. For the test substance, the in vitro irritancy score (IVIS) is 26.04, with a corneal opacity score of 25.333. Based on these results, the benzene, tetrapropylene-, distn. residues, sulfonated, sodium salts cannot be considered an ocular corrosive or severe irritant.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Nov 2017 - 30 Nov 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes
Controls:
yes, concurrent positive control
yes, concurrent negative control
Duration of treatment / exposure:
Two test tissues per test article will be exposed for 30 +/- minutes.
Duration of post- treatment incubation (in vitro):
incubated for 120 +/- 15 minutes
Details on study design:
Pre-Incubation:
EpiOcular™ tissues will be placed in six-well plates containing warmed assay media and will be equilibrated in a humidified 37±1oC, 5 ±1% CO2 incubator for at least one hour. The media will then be changed and the tissues incubated overnight (16-24 hours).
Any tissues not being incubated the same day will be allowed to re-equilibrate at 37±1oC, 5±1% CO2 and will be stored at approximately 2-8oC.

Pre-Treatment:
After the overnight incubation, the tissues will be moistened with 20 μl of Phosphate-Buffered Saline (PBS) and incubated at 37 ±1oC, 5±1% CO2 for 30 ± 2 minutes.

Dosing:
Liquids: 50 μl of a liquid test article will be applied topically to duplicate tissues and incubated at 37±1oC, 5±1% CO2 for 30 ± 2 minutes.
Viscous, waxy, resinous and gel-like test articles with unclear physical state should be incubated at 37±1oC for 15±1 minutes before deciding which treatment protocol to use. If such test articles become pipettable after this incubation period (using a positive displacement pipette, if necessary), they should be treated as liquids and should be applied to the tissues directly from the water bath (at 37±1oC), otherwise they should be treated as solids. Applicator pins inverted onto the tissue may be used to provide a reproducible, even means of application.

Rinse/Soak:
After dosing and incubation, the tissues will be thoroughly rinsed with PBS and soaked in 5 ml of room-temperature assay medium in a 12-well plate for the appropriate amount of time.
Liquids: Tissues will be soaked for 12 ± 2 minutes.

Post-Treatment Incubation:
Tissues will be incubated in 1 ml fresh assay medium in a humidified 37±1oC, 5±1% CO2 incubator.
Liquids will be incubated for 120 ± 15 minutes.

Incubation in MTT:
The tissues will be incubated with 300 μl of 1 mg/ml MTT in DMEM for 3 hours ± 10 minutes at 37±1oC, 5±1% CO2
Tissues for Colorant Controls and Killed Controls for Test articles that are both MTT Reducers and Colorants will be incubated in assay media instead of MTT during the MTT incubation period.

MTT Extraction:
Following the three-hour MTT incubation period, each tissue will be removed individually and gently rinsed with PBS to remove any residual MTT solution.
For liquid test articles, the tissues will be immersed in 2.0 ml of extractant solution per well in a 24-well plate, completely covering the EpiOcular™ tissue (i.e., extraction will occur through both the top and bottom of the insert).
The extraction plate will be covered and sealed to reduce evaporation of extractant.

Extraction Conditions:
The extraction will be allowed to proceed overnight at room temperature in the dark.
Alternatively, the extraction can proceed for at least two hours, with shaking, at room temperature.

Decant Extractant:
Tissues immersed in extractant solution in a 24-well plate: After the extraction period is complete, the liquid within each tissue insert will be decanted back into the well from which it was taken, i.e., the solution will be mixed with the extractant in the well.
Tissues extracted in six-well plate: No liquid is decanted from the tissue insert.
The tissue inserts will then be discarded.

Transferring to 96-Well Plate:
Two 200 μl aliquots from each well of the extraction plate(s) will be pipetted into a 96-well microtiter plate.
Note: If a 96-well plate reader is not available, any visible spectrophotometer will be used to determine optical density of the extracted samples.

Measuring Optical Density:
The optical density of the extracted samples will be determined at a single wavelength of 570 nm and using eight 200 μl aliquots of the Extractant as blanks.
Irritation parameter:
other: % viability measured by Thiazolyl blue tetrazolium bromide (MMT) reduction
Run / experiment:
Mean
Value:
90.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100% viability
Positive controls validity:
valid
Remarks:
44.4% viability
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The test article provided by Advanced Testing Laboratory was tested using the MatTek EpiOcular™ Eye Irritation Test (EIT). The R-squared value calculated for the plate reader linearity check was 0.9999, which met the acceptance criterion of greater than 0.999. All other quality controls passed the acceptance criteria.
Interpretation of results:
GHS criteria not met
Conclusions:
MatTek EpiOcular™ tissue samples were treated in duplicate with the test article, negative control and positive control for 60 minutes. Following treatment and subsequent incubation time, the viability of the tissues was determined using thiazolyl blue tetrazolium bromide (MTT) uptake and reduction. The absorbance of each sample was measured at 570 nm. The viability was then expressed as a percent of negative control values. Mean tissue viability was determined to be 90.7% for the test material, 44.4% for the positive control, and 100% for the negative control. Based on this benzene, tetrapropylene-, distn. residues, sulfonated, sodium saltswas concluded to be a non-ocular irritant.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

SKIN IRRITATION:

Benzene, tetrapropylene-, distn. residues, sulfonated, sodium salt was determined to be non-corrosive in the Corrositex® assay (OECD 439). After confirming the study material is compatible with the assay, a 10% solution of the test material in water was categorized by pH, falling into Corrositex® Category 2. Thus, breakthrough cut-off times of Category 2 were used to determine corrosivity. The test article (0.5 mL) was added to each of four Corrositex® test vials containing macromolecular biobarriers and the times required for the test article to permeate or destroy the biobarriers were recorded. The average breakthrough time for the four test material samples was >85.21 minutes, which exceeds the >60 mins required to be considered non-corrosive. The positive and negative controls (1.0 N sodium hydroxide and 1% citric acid, respectively) were considered valid.

To predict dermal irritation potential, MatTek EpiDerm tissue samples were treated with 30 μl of benzene, tetrapropylene-, distn. residues, sulfonated, sodium salt, phosphate buffered saline [PBS] (negative control), or 5% sodium dodecyl sulfate [SDS] solution (positive control) according to OECD 435. The samples were then incubated in 300 μl methyl thiazole tetrazolium (MTT) solution (1 mg/ml MTT in DMEM) followed by 2.0 mL extractant solution (isopropanol). Mean tissue viability was determined to be 41.6% for the test material, 3.3% for the positive control, and 100% for the negative control. Based on this,benzene, tetrapropylene-, distn. residues, sulfonated, sodium salts was concluded to be a skin irritant Category 2. These in vitro results are supported by a rabbit study conducted under OECD 404 on the read across substance benzenesulfonic acid, methyl-, mono C20 -24 -branched alkyl derivs., calcium salt (Glaza, 1998).

 

Effects on skin irritation/corrosion: Irritating

 

EYE IRRITATION:

In vitro eye irritation studies following OECD guidelines 437 (BCOP-Bovine Corneal Opacity and Permeability Test) and 492 (Reconstructed Human Cornea-like Epithelium Test) were conducted on benzene, tetrapropylene-, distn. residues, sulfonated, sodium salt.

To determine the potential for ocular irritation using an alternative to the Draize methodology, a study based on the methodology described in the current OECD Guideline for the Testing of Chemicals No. 437 was conducted. Three bovine corneas per group were dosed with 0.75 ml of benzene, tetrapropylene-, distn. residues, sulfonated, sodium salt, Minimal Essential Media (MEM, negative control), or 100% ethanol (positive control). Following a 10-minute exposure for each group of dosed corneas, opacity measurements and sodium fluorescein permeability were determined. The MEM solution is then removed from the anterior chamber and replaced with 1.0 ml of 0.4% sodium fluorescein solution. After 90 min the fluid from the posterior chamber is removed and the amount of dye that passed through the cornea is measured as the optical density at 490 nm by a plate reader. For the test substance, the in vitro irritancy score (IVIS) is 26.4 with a corneal opacity core of 25.333. Based on these results, benzene, tetrapropylene-, distn. residues, sulfonated, sodium salt cannot be considered an ocular corrosive or severe irritant.

Tissue samples were treated according to EpiOcular Irritation Test (OECD 492) guidelines to determine its irritation potential. Duplicate tissue samples were treated with benzene, tetrapropylene-, distn. residues, sulfonated, sodium salt, negative and positive control samples for 30 minutes. Following treatment and subsequent incubation time, the viability of the tissues was determined using thiazolyl blue tetrazolium bromide (MTT) uptake and reduction. The absorbance of each sample was measured at 570 nm. The viability was then expressed as a percent of negative control values. Mean viability results for benzene, tetrapropylene-, distn. residues, sulfonated, sodium salt, negative and positive controls were 90.7, 100, and 44.4%, respectively. These findings lead to the classification of benzene, tetrapropylene-, distn. residues, sulfonated, sodium salt as a Non-Irritating to the eye.

This in vitro eye non-irritation potential was corroborated with in vivo studies on the read across source substance calcium sulfonate (CAS 70024-69-0), evaluated in accordance with EPA OPPTS 870.2400 (Kern, 1999). A group of six albino rabbits (3 per sex) was used for the study. Each animal received a single, unwashed exposure of 0.1 mL of the test article instilled into the lower conjunctival sac of the right eye. The eyelid was held closed for approximately one second and released. The left eye was manipulated in a similar manner as the right eye and served as a contralateral control. Observations for eye irritation were made at 1, 24, 48 and 72 hours following dosing and on day 4 in accordance with the method of Draize. One animal had clear discharge from the eye, one-hour post dosing. There were no deaths or changes in bodyweight throughout the study. Mean 24, 48, and 72 hour corneal and iris scores were 0 for all animals tested. The conjunctival erythema scores were 1.3, 1, and 1, respectively and were all reversible within 7 days. The chemosis scores were 0.3, 1.3, 0.3, 0, and 1, respectively. In this study, the test substance is not considered an eye irritant.

A second eye irritation study on the read across source substance calcium sulfonate (CAS 70024-69-0) (Swan, 1972) administered 0.1 mL of test substance into one eye (the other eye served as a control) of adult New Zealand white rabbits (3/sex). The test material was not washed from the eyes. Eyes were scored for corneal, iritis and conjunctival effects 24, 48 and 72 hours following exposure in accordance with the method of Draize. Mean scores at 24, 48 and 72 hours for each animal for corneal opacity and iritis was 0 for all animals. Mean scores for conjunctival redness were 2, 2, 1.3, 1.3, 1 and 1, respectively. Mean scores for chemosis were 2, 1.7, 0.7, 1, 0.3 and 1, respectively. As the scores for conjunctival redness and oedema were less than 2 in 4 animals, this substance is not irritating to rabbit eyes.

In a supporting eye irritation screening study (Buehler, 1990) on the read across source substance calcium sulfonate (CAS 70024-69-0) administered to rabbits (1 male and 1 female) a single, exposure of 0.1 mL of the test article instilled into one eye. The other eye served as a control. The test material was not washed from the eyes. Animals were observed at 1, 24, 48, 72 hours in accordance with the method of Draize. Mean scores at 24, 48 and 72 hours for each animal for corneal opacity and iritis were 0 for all animals. Mean scores for conjunctival redness was 2 in both animals. Conjunctival oedema scores were 2.0 in one animal and 2.33 for the other. In the latter animal the conjunctival oedema decreased from a score of 3 at 24 hours to a score of 2 at 72 hours indicating the effects being reversible. However, because only 2 animals were used, and observations were terminated at 72 hours rather than the normal 21 days, this study cannot be used to determine classification and labelling.

In a definitive supporting study on the read across source substance calcium sulfonate (CAS 70024 -69 -0), that was conducted following the screening study of Buehler (1990), six animals (3 male and 3 female animals) were used to more fully evaluate the eye irritation potential of the calcium sulfonate read across substance, CAS 70024-69-0 (Buehler, 1991). Each animal received a single, exposure of 0.1 mL of the test article instilled into one eye. The other eye served as a control. The test material was not washed from the eyes. Animals were observed at 1, 24, 48, 72 hours in accordance with the method of Draize. Mean scores at 24, 48 and 72 hours for each animal for corneal opacity and iritis was 0 for all animals. Mean scores for conjunctival redness were 1, 0.7, 0.7, 0.3, 0.3 and 1, respectively. Mean scores for chemosis were 1.3, 0.7, 0, 0.7, 0.7 and 1.7, respectively. Except for the 1.7 value for chemosis in animal 6, all other signs were fully reversible within 72 hours. The mean scores for all animals for conjunctival redness and oedema were less than 2. Therefore, this read across source substance is not irritating to rabbit eyes.

 

Effects on eye irritation/corrosion: Non-Irritating


Justification for classification or non-classification

SKIN IRRITATION:

In vitro skin corrosion and irritation studies of sufficient quality and tested in accordance with standard methodology (OECD 435 and 439) showed that benzene, tetrapropylene-, distn. residues, sulfonated, sodium salt was not corrosive, but it was irritating to the skin Category 2. This classification was confirmed by an in vivo skin irritation study on a read across source substance.

EYE IRRITATION:

The eye non-irritation profile of the registered/target substance was determined by in vitro toxicity studies following OECD guidelines 437 (Bovine Corneal Opacity and Permeability Test) and 492 (Reconstructed Human Cornea-like Epithelium Test). This in vitro non-irritation potential was corroborated by several eye irritation in vivo studies of sufficient quality on the read across source substance calcium sulfonate (CAS 70024-69-0) and conducted in accordance with standard methodology. The mean scores in these studies were zero for the cornea and iris. The average scores for conjunctival redness over 24, 48, and 72 hours ranged from 1.0 to 2.0. Overall, mean scores for chemosis and conjunctival redness ranged from 0.3 to 2.0. All adverse effects were fully reversible between 72 hours to 7 days. The cutoff value for eye irritation classification is a positive response of corneal opacity ≥ 3 and/or iritis > 1.5 calculated as the mean scores following grading at 24, 48, and 72 hours after instillation of the test material. Therefore, no classification for eye irritation is proposed for benzene, tetrapropylene-, distn. residues, sulfonated, sodium salt.