reaction products of 1 mole of Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs and 1 mole of cyclohexylamine

Endpoint summary

Description of key information

The dermal and ocular irritant potential of Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.,compds. with cyclohexylamine was assessed using:

- an in vitro skin irritation on Episkin^TMReconstructed human epidermis (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46),

- a bovine corneal opacity and permeability test and an in vitro eye irritation test on epiocular^TMreconstructed human cornea-like epithelium.

Based on these studies, Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.,compds. with cyclohexylamine is considered to be non-irritant for skin and irritant for eyes.

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:

10 February 2017 - 22 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France.

Justification for test system used:

The EpiskinTM model is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum.
Based on the peer review of the results of an inter-laboratory study with the EpiskinTM model, the ECVAM Scientific Advisory Committee (ESAC) endorsed the conclusion that the EpiskinTM model can be used for distinguishing between skin irritant and non-irritant chemicals. The EpiskinTM model is used for hazard identification and the classification of irritation potential within the context of the sequential skin irritation testing strategy (OECD Test Guideline No. 404, 24th April 2002 and Commission Regulation (EC), No. 440/2008, Part B.4, 30 May 2008).

Vehicle:

other: formulation of isopropanol at 1.5% (w/w) in water for injections

Remarks:

The test item was provided as a ready-to-use formulation containing 1.5% (% weight) of isopropanol (IPA).

Details on test system:

The EpiskinTM model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstructed from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra structure and is functionally equivalent to human in vivo epidermis.

Control samples:

yes, concurrent negative control

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Concentration: neat (as supplied)
- Amount(s) applied: (volume or weight with unit): 10 mg (+/- 2 mg)

Duration of treatment / exposure:

Exposure period of 15 minutes, followed by rinsing.

Duration of post-treatment incubation (if applicable):

42-hour recovery period.

Number of replicates:

Three tissues were used for each item.
At the end of the viability assay, formazan level in tissues were assessed in duplicate for each tissue.
The mean OD values (mean OD) were then calculated from the six replicates on each plate.

Type of coverage:

other: not applicable (in vitro)

Preparation of test site:

other: not applicable (in vitro)

Details on study design:

Preliminary tests
Test for direct MTT reduction with the test item
As a test substance may directly reduce MTT, thus mimicking mitochondrial succinate dehydrogenase activity, it is necessary to test the ability of the test item to directly reduce MTT before performing the main test.
To identify any test item interference with the MTT endpoint, the following preliminary test was performed:
- 11 mg (± 1 mg), in order to incubate 10 mg (± 2 mg), of the test item were added to 2 mL of a 0.3 mg/mL freshly prepared MTT solution,
- a negative control was tested concurrently by adding 10 µL of water for injections to 2 mL of a 0.3 mg/mL freshly prepared MTT solution,
- both mixtures were incubated in darkness at +37°C for 3 hours (± 5 minutes).
Then the colour of the solutions obtained was visually evaluated.

Test for the detection of the colouring potential of the test item
The intrinsic colour or the ability of the test item to become coloured in contact with water (simulating a tissue humid environment) was evaluated by adding 11 mg (± 2 mg) of test item to 90 µL of water for injections in a transparent recipient. After 1 hour (± 3 minutes) of mixing, the presence and intensity of the colouration was visually evaluated.

Main test
One 12-well plate was used for each test and control items: one plate for the test item, one plate for the negative control and one plate for the positive control. Each item was applied onto triplicate tissues.

Pre-incubation of the tissues on their day of arrival (Day 0)
A volume of 2 mL of pre-warmed (at +37°C) maintenance medium was added to 3 wells on a 12 well plate (one plate per item).
Each Episkin TM tissue was then transferred into the maintenance medium pre-filled wells (three tissues per plate). The plates were incubated at +37°C, 5% CO2 in a humidified incubator for at least 2 hours (based on literature Assessment of the Effects of Pre-Incubation Time on Irritation Potential Using the EpiSkin® in vitro Irritation Test, C. Blackstock and C. Rope; Skin Forum, Edinburgh 07/2010).

Treatment of tissues (Day 1)
A volume of 2 mL of pre-warmed maintenance medium was added into 3 wells on the 12 well plate for each item, respectively.
Each test or control item was then applied on three tissues for an exposure period of 15 minutes and according to quantities defined in § Test item, vehicle, positive and negative controls administrations. The items were applied topically to the corresponding tissues and gently spread over the epidermal surfaces to ensure uniform covering of the tissues (see § Study plan adherence). The tissues were processed (treatment and rinsing) in the same order and at regular time intervals to ensure each tissue received an equal exposure period.

For the positive control only, the SDS solution was re-spread after 7 minutes (± 1 minute) contact time with a curved spatula to improve the distribution of the SDS for the remainder of the contact period.

The exposure of the tissues to the test and control items was performed at room temperature for 15 minutes (± 1 minute).

Rinsing of tissues and incubation for 42 hours (Day 1)
At the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS. Rinsing was achieved by gently filling and emptying several times each tissue with D PBS to gently remove any residual test or control items. The epidermal surface of the tissues treated with the test item was gently swept with a cotton-bud to remove excess D-PBS (without damaging the epidermis).
The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well and the plates were incubated at +37°C, 5% CO2 in a humidified incubator for 42 hours.

MTT viability assay (Day 3)
Following the 42 hours incubation period, plate containing test item, vehicle and negative control-treated tissues were placed for 15 minutes (± 2 minutes) on a plate shaker to homogenise the released inflammatory mediators in the maintenance medium. Then, 1.6 mL of incubation medium from beneath each tissue were retained in pre-labeled micro tubes, and stored at -20°C until quantification for inflammatory mediators.
Then, 2 mL of a freshly prepared MTT solution (0.3 mg/mL) were added into 3 wells on each 12 well plate. The tissues were then transferred to the MTT filled wells and incubated for 3 hours (± 5 minutes) at +37°C, 5% CO2 in a humidified incubator.

At the end of the MTT incubation period, a total biopsy of the epidermis was made by using the Episkin™ biopsy punch. Each tissue was examined with the naked eye and the degree of MTT staining was evaluated. The epidermis was separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into micro tubes and incubated with 500 µL of acidic isopropanol. Then, to extract the formazan (reduced MTT) from the MTT-loaded tissues, each tube was stored after vortexing at room temperature and protected from light for 4 hours and 2 minutes.

Optical Density measurements (Day 6)
At the end of the formazan extraction period, each tube was vortexed until the solution colour became homogenous.
Each tube was used to fill two wells of a 96-well plate with 200 µL of extract per well. One 96 well plate was used for the negative and positive controls (placed at opposite ends of the plate), and a separate 96-well plate was used for the test item.
For each 96-well plate, the average Optical Density value (OD) of 6 wells containing 200 µL of acidified isopropanol only was used as the blank.

The OD was measured at 570 nm using a plate reader.

The IL-1a concentrations in culture media samples retained from the three negative controls, the three test item-treated and the three vehicle control tissues were analyzed by ELISA.

Irritation / corrosion parameter:

% tissue viability

Remarks:

15 min

Run / experiment:

Mean

Value:

84

Vehicle controls validity:

not applicable

Remarks:

95%

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

6%

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other: Mean IL1a concentration (pg/mL)

Run / experiment:

Main

Value:

10.2

Vehicle controls validity:

not applicable

Remarks:

< 12.4

Negative controls validity:

valid

Remarks:

<7.74

Positive controls validity:

not examined

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- Evaluation of the colouration of tissues at the end of the MTT incubation period
The qualitative evaluation of the MTT staining was performed with the naked eye.
All test item and vehicle-treated tissues appeared blue which was considered indicative for viable tissues.

- Evaluation of the MTT results
All of the acceptance criteria for the negative and positive controls were fulfilled, therefore the study was considered to be valid.

Interpretation of results:

other: non-irritant to the skin

Conclusions:

Under the experimental conditions of this study, the test item is considered to be non-irritant to skin.
According to the results of this study, the classification of the test item should be:
- No Category (UN GHS and Regulation (EC) No. 1272/2008).

Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test item using the Episkin^TM reconstructed human epidermis model. As the test item was provided as a ready-to-use formulation containing 1.5% (% weight) of isopropanol, a formulation of isopropanol at 1.5% (w/w) in water for injections was used as a vehicle control in order to determine possible irritant properties of the test item-related to the isopropanol content.

 

The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46). The study was conducted in compliance with CiToxLAB France standard operating procedures and the principles of Good Laboratory Practice.

 

Methods

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential.

Following the preliminary tests, the skin irritation potential of the test item and the vehicle control were tested in the main test. The test item, the vehicle control and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at +37°C, 5% CO₂ in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay.

Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

In addition, the concentration of the inflammatory mediator IL-1a was evaluated in the culture medium retained following the 42-hour recovery period. This quantification, based on an ELISA assay, was performed since the mean relative viability of the test item-treated tissues was > 50% following the MTT reduction assay.

 

Results

Preliminary tests

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential.

 

Main test

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viabilities of the tissues treated with the test item and with the vehicle control were:

- test item: 84% with a Standard Deviation of 9%,

- vehicle control: 95% with a Standard Deviation of 5%.

 

As all mean viabilities were > 50% after the MTT reduction, the IL-1a concentrations in culture media samples retained from the three negative controls, the three test item and the three vehicle control-treated tissues were analyzed by ELISA:

- test item: the mean IL-1a concentration was 10.2 pg/mL. Due to this value being below 60 pg/mL, the results met the criteria for an in vitro classification as non-irritant to skin,

- vehicle control: the IL-1a concentration value from one tissue was found Below the Limit Of Quantification (< 5.00 pg/mL). Consequently, the mean IL-1a concentration from the three vehicle-treated tissues was not calculated. The IL-1a concentration values from the tissue Nos. 1 and 3 were found < 60 pg/mL (i.e. 12.4 and 6.46 pg/mL, respectively).

Therefore, the results met the criteria for an in vitro classification as non-irritant to skin.

 

Conclusion

Under the experimental conditions of this study, the test item is considered to be non-irritant to skin.

According to the results of this study, the classification of the test item should be:

- No Category (UN GHS and Regulation (EC) No. 1272/2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 February 2017 - 22 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 437 (In vitro eye irritation)

Version / remarks:

26 July 2013

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

- Source: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
- Age at study initiation: as French Authorities avoid the use of any organs from the head of bovines aged more than 12 months, bovine cattle were up to 12 months old (typically, 5 to 8 months old).
- Transport from Supplier to CiToxLAB France: the eyes were transported to CiToxLAB France at
ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts
Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal
surfaces was used for the transport to avoid damage to the corneas.
- Storage of the corneas: as the corneas were not used immediately, they were stored after washing. Each cornea was stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C, for a maximum of 24 hours before use.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

other: Reference item: pre-formulation of isopropanol at 1.5% (w/w) in water for injections diluted at the concentration of 10% (w/v) in water for injections.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 10% in vehicle

RE§FERENCE ITEM
- Amount applied: 750 µL
- Concentration: 10% in vehicle

Duration of treatment / exposure:

Exposure period of 10 minutes (± 30 seconds), followed by rinsing

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

2 hours (± 10 minutes)

Number of animals or in vitro replicates:

3 corneas per item

Details on study design:

EYES SELECTION
The corneas were carefully examined macroscopically before their assembly in the holders, in order to detect the presence of any defects. Any corneas with defects were discarded. The corneas were then mounted in the corneal holders with the endothelial side against the O-ring of the posterior chamber. Each cornea was identified with the corresponding holder number. After pre-incuvbation, corneas that showed any macroscopic defect or an opacity value over 7 were discarded.

TREATMENT
Three corneas from the same series were always processed in the same order at each step. The medium of the anterior chamber was removed and each item was applied onto the epithelium of the cornea for 10 minutes in a water bath at +32°C (± 1°C).

REMOVAL OF TEST SUBSTANCE
The purpose of rinsing was to eliminate as much items as possible, while taking care not to damage the cornea. On completion of the treatment period, the test item formulation and the reference item were removed from the front opening of the anterior chamber (closed-chamber method) and the epithelium was rinsed as follows:
- the anterior chamber was emptied using a metal gavage tube attached to a vacuum pump,
- the corneas were rinsed four times for the test item formulation and three times for the reference item with pre-warmed cMEM containing phenol red (i.e. until the test item formulation had been completely removed from the chamber or until the phenol red was not discoloured). Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.

SCORING SYSTEM
- Opacity:
An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. The average change in opacity for the corneas treated with the vehicle control was calculated and this value was subtracted from the change in opacity for each cornea treated with test item formulation, reference item or positive control to obtain a corrected opacity value (cOPT). When the average change in opacity for the corneas treated with the negative/vehicle control was negative, it is considered equal to 0.
The mean cOPT value of each series of three corneas was calculated from the individual corrected opacity values.

- Permeability:
After the second opacity measurement, the medium of the anterior chamber was removed and the anterior chamber received 1 mL of a fluorescein solution in a water bath at +32°C (± 1°C) for 90 minutes (± 5 minutes). At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube. The corrected OD490 nm (cOD490 nm) value (i.e. permeability) for each cornea treated by test item formulation or positive control was calculated by subtracting the average negative (or vehicle) control cornea OD490 nm value from the original OD490 nm value of each cornea. When the average negative/vehicle control cornea OD490 nm value was negative, it is considered equal to 0.

- Scoring:
In vitro irritancy score (IVIS) = cOPT + (15 x cOD490 nm)

Irritation parameter:

in vitro irritation score

Remarks:

test item

Run / experiment:

mean

Value:

16

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Remarks:

Reference item

Run / experiment:

mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Remarks:

Positive control

Run / experiment:

mean

Value:

50

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS (macroscopic examination):
Vehicle corneas: non
Test item: fluorescein fixation and opacity on each cornea
Reference item: none
Posiive control: incease in thickness, fluorescein fixation and opacity on each cornea

ACCEPTANCE OF RESULTS:
For the validation of an experiment, the following criteria had to be fulfilled:
- the mean In Vitro Irritancy Score (IVIS) of the positive control corneas should fall within two Standard Deviations of the historical mean,
- the mean opacity and mean OD490 nm of the vehicle control corneas should be less than the established upper limit of historical mean.

Interpretation of results:

other: No indication of severe irritation/corrosion

Conclusions:

Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as not requiring classification for eye irritation or serious eye damage (UN GHS No Category). According to OECD Guideline 437, further testing should be conducted for classification and labeling purposes.

Executive summary:

The objective of this study was to evaluate the potential irritant and corrosive properties of the test item to the eye. The Bovine Corneal Opacity and Permeability (BCOP) test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage.

The design of this study was based on the guideline OECD Guideline 437 and the study was performed in compliance with CiToxLAB France standard operating proceduresand with the OECD Principles of Good Laboratory Practice.

 

Method

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C.

Since the test item contained 1.5% isopropanol, a 1.5% (w/w) isopropanol solution was prepared in water for injections, and diluted identically to the test item before use, in order to investigate whether possible corneal damages would be related to the test item itself, or to its content in isopropanol.

A single experiment was performed using three corneas for each treated series (test item formulation, reference item, vehicle control and positive control).

Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer.

The test item, tested at 10% (w/v) in the vehicle (0.9% NaCl), reference item, vehicle and positive controls were evaluated in a single experiment using a treatment time of 10 minutes and the closed-chamber treatment method. At completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed.

The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C.

At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

 

Results

Macroscopic examination

Opacity and fluorescein fixation were observed on the corneas treated with the test item formulation. No finding was noted on the corneas treated with the reference item.

 

In Vitro Irritancy Score

All acceptance criteria were fulfilled. The study was therefore considered as valid.

The mean In Vitro Irritancy Score (IVIS) of the test item formulation-treated corneas was: 16.

The mean In Vitro Irritancy Score (IVIS) of the reference item-treated corneas was: 0.

 

As the IVIS obtained with the reference item was 0, all findings noted on test item formulation-treated corneas were considered to be due to its 98.5% UVCB content, and not to the 1.5% isopropanol content.

 

As the mean IVIS was > 3 and < 55, the eye hazard potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Conclusion

Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as not requiring classification for eye irritation or serious eye damage (UN GHS No Category). According to OECD Guideline 437, further testing should be conducted for classification and labeling purposes.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 August 2017 - 08 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: reconstructed human Cornea-like epithelium (tissues)

Details on test animals or tissues and environmental conditions:

Cell type: non-transformed keratinocytes
Cell supplier: MatTek, Bratislava, Slovak Republic.

Details on test system and justification for its use:
The EpiOcularTM model consists of an airlifted, living, multilayered ocular tissue construction (surface 0.60 cm2), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a non-keratinized epithelium which models the cornea epithelium with progressively stratified, but not cornified cells. The cells are cultured in proprietary serum-free culture media, which induces corneal differentiation and the formation of the organotypic 3D cornea-like model. The 3D tissue consists of highly organized cell layers similar to that found in the cornea. The model features a normal ultra-structure and is functionally equivalent to human in vivo tissue.
Its use for eye irritation testing involves a topical application of test items on the surface of the cornea epithelial construct for different exposure incubations.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Concentration: neat (as supplied),
- Amount applied: 50 mg (± 2 mg) per tissue, taking care to spread it over the whole tissue surface area without damaging the tissue sample.

Duration of treatment / exposure:

Exposure period of 6 hours (+/- 15 minutes) at +37°C, 5% CO2 in a humidified incubator, followed by rinsing.

Duration of post- treatment incubation (in vitro):

Post-incubation period of 18 hours (± 15 minutes) at +37°C, 5% CO2 in a humidified incubator.

Number of animals or in vitro replicates:

Test item, negative and positive controls were applied on duplicate tissues.
At the end of the viability assay, formazan level in tissues was assessed in duplicate for each tissue.
The mean OD values were then calculated from the four replicates for each item.

Details on study design:

PRELIMINARY TEST
* Test for direct MTT reduction with the test item
As a test item may directly reduce MTT, thus mimicking mitochondrial succinate dehydrogenase activity, it is necessary to test the ability of the test item to directly reduce MTT before performing the main test. To identify any test item interference with the MTT endpoint, the following preliminary test was performed:
- 50 mg (± 2 mg) of the test item were added to 1 mL of a 1.0 mg/mL freshly prepared MTT solution,
- a negative control was tested concurrently by adding 50 µL of sterile deionized water to 1 mL of a 1.0 mg/mL freshly prepared MTT solution,
- both mixtures were incubated in darkness at +37°C for 3 hours (± 10 minutes).
Then the colour of the both solutions obtained was evaluated.

* Test for the detection of the colouring potential of the test item
As a test item may be coloured or become coloured in contact with water and/or isopropanol, it is necessary to test its potential interference with the MTT determination in these two conditions. The maximum amount of test item, 50 mg (± 2 mg), was added to 1 mL of water and incubated for at least one hour at +37°C, 5% CO2 and 2 mL of isopropanol, incubated in a 6-well plate and placed on an orbital plate shaker for 2 to 3 hours at room temperature. After that, the presence and intensity of the colouration were evaluated.


MAIN TEST
One 6-well plate was used for the test item-treated tissues. Positive and negative controls were placed on separate 6-well plates (one plate for each).
Test item, negative and positive controls were applied on duplicate tissues.

PRE-INCUBATION OF THE TISSUES
As the tissues were shipped before the day prior the treatment, tissues were stored between +2°C and +8°C, prior their pre-incubation. The tissues were equilibrated (in the 24-well shipping container) to room temperature for at least 15 minutes. The underside of each tissue was inspected for air bubbles between the agarose gel and the insert. Tissues with air bubbles under the insert covering greater than 50% of the insert area were not used. Any unusual observation was noted separately. The plastic bag containing the 24-well plate shipping container was disinfected by wiping with 70% ethanol-soaked tissue paper. Each 24-well shipping container was then removed from its plastic bag under sterile conditions. A volume of 1 mL assay medium pre-warmed (+37°C) was added to 2 wells per pre-labelled 6-well plate. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plate and pre-incubated at +37°C, 5% CO2 in a humidified incubator for 1 hour. After the pre-incubation period, the assay medium was removed and replaced by 1 mL of fresh assay medium before incubation overnight (16-24h) at +37°C, 5% CO2 in a humidified incubator. Each 6-well plate was labelled with the test item or control codes.

TREATMENT
Following the pre-incubation period, the tissues were pre-wetted with 20 µL of D-PBS. Each insert was tapped to ensure that the entire tissue surface was wetted. The tissues were then incubated at +37°C, 5% CO2 in a humidified incubator for 30 (± 2) minutes. As the test item was solid, the insert was removed from the medium and placed onto a sterile surface (e.g. the lid of a microtiter plate) to avoid that test item was spilled into the assay medium under the tissue insert. The test item was then applied by pouring it onto the tissue surface so that the surface was completely covered by the test item. The test item, negative and positive controls were applied topically on each designated tissue, and gently spread onto the epithelium surfaces to ensure uniform covering of the tissues.
Inserts were then tapped on the wall of the plate to ensure that the items were applied evenly to the surface of each tissue. The tissues were placed back into the assay medium after treatment with the test item.
All tissues (test item, negative and positive controls) were incubated at +37°C, 5% CO2 in a humidified incubator for 6 hours (± 15 minutes).The tissues were processed (treatment and rinsing) in the same order and at regular time-intervals to ensure each tissue receives an equal exposure period.

RINSING
At the end of the treatment period, each tissue was removed from the well of the treatment plate and rinsed to gently remove any residual test or control items. A set of three clean beakers containing a minimum of 100 mL each of D-PBS was used per test or control item. The test or control items were firstly removed from the tissue surface by tapping upside down each insert onto a clean absorbent paper. The tissues were then dipped into the first beaker of D-PBS, swirled in a circular motion during approximately 2 seconds, lifted out and decanted back into the beaker. This process was performed three times per beaker.
Any remaining liquid was decanted onto an absorbent paper.

POST-SOAK AND POST-INCUBATION
The rinsed tissues were transferred to new wells of a pre-labelled 12-well plate containing 5 mL of assay medium pre-warmed at room temperature. This incubation in assay medium was intended to remove any test article from the tissue. Each tissue was thus incubated for 25 minutes (± 2 minutes) at room temperature to remove any solid test item or negative and positive controls from the tissue.
At the end of the post-soak immersion, each insert was blotted on absorbent material and transferred to appropriate well of the pre-labelled 6-well plate containing 1 mL of assay medium. The tissues were then incubated for 18 hours (± 15 minutes) at +37°C, 5% CO2 in a humidified incubator for test item, negative and positive controls.

MTT VIABILITY ASSAY
Following the post-treatment incubation, a volume of 0.3 mL of a freshly prepared MTT solution (1.0 mg/mL) was added into new wells of pre-labelled 24-well plates.
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent paper. The tissues were then transferred to the MTT pre-filled wells and incubated for 3 hours (± 10 minutes) at +37°C, 5% CO2 in a humidified incubator. At the end of the 3-hour incubation period, the underside of each tissue was blotted on absorbent paper to dry. Each tissue was examined with the naked eye and the degree of MTT staining was evaluated.
For the negative and positive control-treated tissues, the inserts were transferred to new wells of the 24-well plate containing 2 mL of isopropanol per well so that isopropanol was flowing into the insert on the tissue surface. As the test item was a non-colourant solid, the inserts were transferred to a 6-well plate containing 2 mL of isopropanol in each well so that no isopropanol was flowing into the insert. This avoided any potential contamination of the isopropanol solution with any test item that may have remained on the tissue.
Plates were surrounded with parafilm to prevent evaporation. Formazan extraction was performed during 2 to 3 hours at room temperature by placing the plates on an orbital plate shaker.

OPTICAL DENSITY MEASUREMENTS
At the end of the formazan extraction period, tissues (test item, negative and positive control-treated tissues) were not pierced. The extract solution was mixed using a pipette and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
One 96-well plate was used for the negative and positive controls (placed at opposite end of the plate), and a separate 96-well plate was used for test item-treated tissues.
For each 96-well plate, the average Optical Density value (OD) of 4 wells containing 200 µL of isopropanol only was used as the blank.
The OD was measured at 570 nm using a plate reader.

DATA ANALYSIS
As the test item was found in the preliminary test neither to have any colouring potential, nor any direct MTT reducing properties, no additional controls were run during the main test. Therefore, the mean blank OD value (mean ODblank) was calculated from the four replicates. Then, the mean ODblank was subtracted from each OD value and the corrected mean OD values (mean cOD) of the two aliquots were calculated for each tissue. The corrected mean OD of the two negative controls-treated tissues (mean cODNC) was set to 100% viability and was used as a reference.
For the tissues treated with the test item or negative or positive controls, the relative viabilities for each tissue were expressed as percentages of the reference viability and were calculated as follows:
TI relative viability (%) = (cODTI or NC or PC / mean cODNC) x 100

With: cODTI = corrected OD of each tissue treated with test item.
cODNC= corrected OD of each tissue treated with negative control.
cODPC= corrected OD of each tissue treated with positive control.

Irritation parameter:

other: % cell viability

Run / experiment:

mean

Value:

1

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

27%

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- the mean cOD of the negative controls should be between 0.8 and 2.5,
- the relative mean viability of the positive control should be < 50% of the relative mean viability of the negative control,
- the difference of viability between the two tissue replicate should be < 20%.

EVALUATION OF THE COLORATION OF TISSUES AT THE END OF THE MTT INCUBATION PERIOD
The qualitative evaluation of the MTT staining was performed with the naked eye.
All test item-treated tissues appeared white (with a yellow coloration of both tissues) which was considered to be indicative of non-viable tissue.

EVALUATION OF MTT RESULTS
All of the acceptance criteria for the negative and positive controls were fulfilled (Table 2), therefore the study was considered to be valid.

Irritant / corrosive response data:

The relative mean viability of the tissues treated with the test item was 1% with a difference of 0% between duplicate tissues.
As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response.

Interpretation of results:

other: Between Category 1 (H318) and Category 2 (H319)

Conclusions:

Under the experimental conditions of this study, the test item is considered to be irritant to Reconstructed human Cornea-like Epithelium.
According to the results of this study, the classification of the test item should be Category 1/Category 2 (GHS 2015) and Category 1 (H318)/Category 2 (H319) (Regulation (EC) No. 1272/2008).

Executive summary:

The purpose of this study was to predict the acute eye irritation potential of the test item by measurement of its cytotoxic effect on the EpiOcular^TMcornea epithelial model.

The design of this study was based on the OECD Guideline 492 and the study was performedin compliance with CiToxLAB France standard operating proceduresand with the OECD Principles of Good Laboratory Practice.

 

Methods

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential.

Following the preliminary tests, the eye irritation potential of the test item was assessed in the main test. The test item and both negative and positive controls were applied topically on duplicate tissues and incubated at +37°C for 6 hours. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 25 minutes at room temperature to remove any remaining test item from the tissue, blotted on absorbent material, and then incubated for another 18 hours at, 5% CO₂in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay.

Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (as reference viability).

 

Results

Preliminary tests

In the preliminary tests, the test item was neither found to have direct MTT reducing properties, nor colouring potential.

 

Main test

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

The relative mean viability of the tissues treated with the test item was 1% with a difference of 0% between duplicate tissues.

As the mean viability was < 60% after the MTT reduction,the results met the criteria for an irritant response.

 

Conclusion

Under the experimental conditions of this study, the test item is considered to be irritant to Reconstructed human Cornea-like Epithelium.

According to the results of this study, the classification of the test item should be Category 1/Category 2 (GHS 2015) and Category 1 (H318)/Category 2 (H319) (Regulation (EC) No. 1272/2008).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

An in vitro skin irritation test was performed to evaluate the skin irritation potential of the test item, Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with cyclohexylamine, using the EpiskinTM reconstructed human epidermis model. As the test item was provided as a ready-to-use formulation containing 1.5% (% weight) of isopropanol, a formulation of isopropanol at 1.5% (w/w) in water for injections was used as a vehicle control in order to determine possible irritant properties of the test item-related to the isopropanol content.

 The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46).

The test item, the vehicle control and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, the cell viability was then assessed. In addition, the concentration of the inflammatory mediator IL-1a was evaluated on an Elisa assay in the culture medium retained following the 42-hour recovery period since the mean relative viability of the test item-treated tissues was > 50% following the MTT reduction assay.

As the mean IL-1a concentration for test item was below 60 pg/mL, the results met the criteria for anin vitroclassification as non-irritant to skin.

Eye irritation

A bovine corneal opacity and permeability testThe objective of this study was performed to evaluate the potential irritant and corrosive properties of the test item, Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with cyclohexylamine, to the eye. The design of this study was based on the OECD Guideline 437.

Since the test item formulation contained 1.5% isopropanol, a 1.5% (w/w) isopropanol solution was prepared in water for injections, and diluted identically to the test item before use, in order to investigate whether possible corneal damages would be related to the test item itself, or to its content in isopropanol.

A single experiment was performed using three corneas for each treated series (test item formulation, reference item, vehicle control and positive control).

Opacity and fluorescein fixation were observed on the corneas treated with the test item formulation. No finding was noted on the corneas treated with the reference item.

As the mean In Vitro Irritancy Score (IVIS) was > 3 and < 55, the eye hazard potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (Category 1/Regulation (EC) No. 1272/2008) or as a test chemical not requiring classification for eye irritation or serious eye damage (Regulation (EC) No. 1272/2008).

Thus, a second in vitro study, the EpiOcularTM cornea epithelial model was performed.

The purpose of this study was to predict the eye hazard potential of the test item by measurement of its cytotoxic effect. The design of this study was based on the OECD Guideline 492 accordingly to OECD Principles of Good Laboratory Practice.

The test item and both negative and positive controls were applied topically on duplicate tissues and incubated at +37°C for 6 hours. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 25 minutes at room temperature to remove any remaining test item from the tissue, blotted on absorbent material, and then incubated for another 18 hours at 5% CO2in a humidified incubator.

The cell viability was then assessed by means of the colourimetric MTT reduction assay. Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (as reference viability). As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. All acceptance criteria for the negative and positive controls were fulfilled and the study was therefore considered to be valid.

Under the experimental conditions of this study, the test item is considered to be irritant to Reconstructed human Cornea-like Epithelium. According to the results of this study, the classification of the test item should be Category 1 (H318) or Category 2 (H319) (Regulation (EC) No. 1272/2008).

Taking into account of the overall results, the test item is considered to be classified eye irritant category 2 H319 (Regulation (EC) No. 1272/2008)

Justification for classification or non-classification

According to available data, the test item, Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with cyclohexylamine, is considered to be:

- non-irritant to skin;

- eye irritant category 2, H319