Sulfonic acids, shale-oil, sodium salts

Endpoint summary

Description of key information

The EPISKIN Reconstructioned Human Epidermis Model and the Bovine Corneal Opacity and Permeability (BCOP) Assay have been used to determine the skin irritation potential and the eye irritation potential of Sulfonic acids, shale oil , sodium salts.

Skin Irritation Potential

The purpose of the test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours.

The relative mean viability of the test item treated tissues was 63.5% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied. The test item was classified as non-irritant.

Eye Irritation Potential

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage. The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The In Vitro Irritancy Scores (IVIS) are 25.1 (Test Item), 0.5 (Negative Control) and 51.6 (Positive Control) respectively. Accordinly, the test item is neither "non irritating" nor "seriously eye damaging". Nonetheless, no prediction of eye irritation can be made, i. e. no distinction between Cat. 2A (irritating) and Cat. 2B (mildly irritating) is possible but (since IVIS 3 < 21.5 < 55) Category 2 (irritating to eyes) must be assumed.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

initiated: 2016-08-22, experimental: 2016-11-08 to 2016-11-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

recommended study/method

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Deviations:

yes

Remarks:

In the initial run of this test, an additional procedure using viable color correction tissues was performed to measure any potential interference. This deviation was considered to have not affected the integrity or validity of the study.

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2009

Deviations:

yes

Remarks:

In the initial run of this test, an additional procedure using viable color correction tissues was performed to measure any potential interference. This deviation was considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

MHRA, date of issue: 28/10/2016

Specific details on test material used for the study:

technical grade: aqueous solution (31% dry matter in water)

Test system:

human skin model

Source species:

other: reconstructed human epidermis model

Cell type:

other: adult human-derived epidermal keratinocytes

Cell source:

other: seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen

Source strain:

not specified

Details on animal used as source of test system:

no animals used in this study

Justification for test system used:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay.

Details on test system:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 02 November 2016
EpiSkinTM Tissues (0.38cm2) lot number: 16-EKIN-045
Maintenance Medium lot number: 16-MAIN3-075
Assay Medium lot number: 16-ESSC-048


PRE-TEST PROCEDURE: ASSESSMENT OF DIRECT TEST ITEM REDUCTION OF MTT
MTT Salt Metabolism, Cell Viability Assay:
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.

Test for Direct MTT Reduction:
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure: 10 μL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue or purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

Assessment of Color Interference with the MTT endpoint:
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. 10 μL of test item was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made. The test item was considered to have the potential to cause color interference with the MTT endpoint. Therefore an additional procedure was performed using viable tissues to assess for the possibility of color interference. 10 μL of the test item was applied to three viable tissues in parallel to the main test with the exception that the MTT incubation period was replaced by incubation with maintenance medium (therefore without MTT). Three viable tissues were also used for negative control purposes which remained untreated. The optical density measurements from these tissues were then assessed for possible quantitative correction of the results.

MAIN TEST
Application of Test Item and Rinsing (Day 1):
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3):
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination. 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6):
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

Control samples:

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

MAIN TEST
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate.
10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes.

Duration of post-treatment incubation (if applicable):

The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item 1

Value:

54

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item 2

Value:

72.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item 3

Value:

63.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item, relative mean

Value:

63.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

POSITIVE CONTROL ITEM
The relative mean tissue viability for the positive control treated tissues was 7.6% relative to the negative control treated tissues and the standard deviation value of the viability was 0.4%. The positive control acceptance criteria were therefore satisfied.

NEGATIVE CONTROL ITEM
The mean OD562 for the negative control treated tissues was 0.813 and the standard deviation value of the viability was 0.097%. The negative control acceptance criteria were therefore satisfied.

TEST ITEM
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 9.3%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant.

Executive summary:

Study / Method

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours.

Result

The relative mean viability of the test item treated tissues was 63.5% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

Conclusion

The quality criteria required for acceptance of results in the test were satisfied. The test item was classified as non-irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

initiated: 2016-08-22, experimental: 2016-12-13 to 2016-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

recommended study/method

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

MHRA, date of issue: 28/10/2016

Specific details on test material used for the study:

technical grade: aqueous solution (31% dry matter in water)

Species:

cattle

Strain:

not specified

Remarks:

eyes from adult cattle (typically 12 to 60 months old)

Details on test animals or tissues and environmental conditions:

SOURE OF BOVINE EYES
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TREATMENT OF CORNEAS
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas.

APPLICATION OF SODIUM FLUORESCEIN
The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL).

Duration of treatment / exposure:

TREATMENT OF CORNEAS
The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.

APPLICATION OF SODIUM FLUORESCEIN
The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Duration of post- treatment incubation (in vitro):

The condition of the cornea was visually assessed post treatment and post incubation (see Appendix 2).

Number of animals or in vitro replicates:

CORNEA NUMBER
- Negative Control: 3
- Positive Control: 3
- Test Item: 3

Details on study design:

PREOARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

SELECTION OF CORNEAS AND OPACITY READING
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer (see Annex 1). The average opacity for all corneas was calculated. Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

TREATMENT OF CORNEAS
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes. At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

APPLICATION OF FLUORESCEIN
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

PERMEABILITY DETERMINATIONS
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

HISTOPATHOLOGY
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

DATA EVALUATION
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

OPACITY MEASUREMENT
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

PERMEABILITY MEASUREMENT
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

IN VITRO IRRITANCY SCORE
The following formula was used to determine the In Vitro Irritancy Score: In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value). Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

VISUAL OBSERVATION
The condition of the cornea was visually assessed post treatment and post incubation.

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Item

Value:

25.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made

Other effects / acceptance of results:

DATA INTERPERTATION
The test item was classified according to the following prediction model:
IVIS Classification
≤ 3 No category. Not requiring classification to UN GHS or EU CLP
> 3; ≤55 No prediction of eye irritation can be made
> 55 Category 1. UN GHS or EU CLP Causes serious eye damage

CRITERIA FOR AN ACCAPTABEL TEST
For an acceptable test the following positive control criterion should be achieved: Neat ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during 2015 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 27.2 to 53.4.
For an acceptable test the following negative control criteria should be achieved: 0.9% w/v sodium chloride solution was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2015 for bovine corneas treated with the respective negative control. When testing liquids the negative control limit for opacity should be ≤ 2.8 and for permeability ≤ 0.115.

Interpretation of results:

other: No prediction of eye irritation can be made

Remarks:

Since IVIS 3 < 21.5 < 55 Category 2 (irritating to eyes) must be assumed.

Conclusions:

No prediction of eye irritation can be made. Since IVIS 3 < 21.5 < 55 Category 2 (irritating to eyes) must be assumed. However, no distinction between Cat. 2A (irritating) and Cat. 2B (mildly irritating) is possible.

Executive summary:

Study / Method

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage. The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

Result

The In Vitro irritancy scores are summarized as follows:

Treatment In Vitro Irritancy Score (IVIS)

Test Item 25.1

Negative Control 0.5

Positive Control 51.6

Conclusion

No prediction of eye irritation can be made. Since IVIS 3 < 21.5 < 55 Category 2 (irritating to eyes) must be assumed. However, no distinction between Cat. 2A (irritating) and Cat. 2B (mildly irritating) is possible.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Read the Description of key information / Eye Irritation potential to better understand the Eye irritation / Endpoint conclusion.

Justification for classification or non-classification

Skin irritation potential: The quality criteria required for acceptance of results in the test were satisfied. The test item was classified as non-irritant.

Eye irritation potential: The positive control acceptance criterion was satisfied. The negative control acceptance criteria was satisfied. No prediction of eye damage can be made, however, irritation to eyes (Category 2) must be assumed.