Methyl toluene-4-sulphonate

Endpoint summary

Description of key information

In vitro skin corrosivity

The results indicate that the test item is non-corrosive to the skin.

In vitro skin irritation

The results indicate that the test item is irritant to skin.

In vitro eye irritation

Based on the in vitro eye irritation in the isolated chicken eyes test with PTSM, the test item is non-irritant.

Experience of use

An incident during use resulted in exposure to a worker that in turn resulted in an adverse response in excess of the response predicted by the in vitro assessments. Following an accidental exposure to an unconfirmed amount of the substance, a worker wearing work wear suffered first and second degree burns. As a precautionary measure, the substance is therefore classified as corrosive.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 June 2017 to 02 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142 (31 May 2008)

Deviations:

yes

Remarks:

Deionised water was used instead of distilled water in the check test (colouring potential of test-item). However, this fact had no impact on the results or integrity of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

OECD Guidelines for the Testing of Chemicals, No. 431, (29 July 2016) “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method”

Deviations:

yes

Remarks:

Deionised water was used instead of distilled water in the check test (colouring potential of test-item). However, this fact had no impact on the results or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: three-dimensional human epidermis model.

Source strain:

other: Batch No.: 17-EKIN-022

Details on animal used as source of test system:

EPISKINTM(SM) (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-022, Expiry Date: 05 June 2017) is a three-dimensional human epidermis model. Adult human derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Justification for test system used:

The EPISKINTM(SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKINTM(SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkinTM(SM) Test Kits used in the present study)..

Kit Contents
Units: EPISKINTM(SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EPISKINTM(SM) biopsy punch for easy sampling of epidermis
Medium:
A flask of sterile “Maintenance Medium” (Batch No.: 17 MAIN3 022; Exp. Date: 07 June 2017)
A flask of sterile “Assay Medium” (Batch No.: 17 ESSC 021; Exp. Date: 07 June 2017)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

50 μL of test item was applied evenly to the epidermal surface of each of two test units.
50 μL of physiological saline was added to each of the two negative control skin units.
50 μL of glacial acetic acid was added to each of the two positive control skin units.

Duration of treatment / exposure:

Single treatment.

Duration of post-treatment incubation (if applicable):

The plates with the treated epidermis units were incubated for 4 hours

Number of replicates:

Two replicates per test item were used.
Two negative controls and two positive controls were also run in this assay.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item-Mean

Value:

96.6

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:

As the test item was colourless and it had no colouring potential, no additional controls were used for the non-specific OD evaluation.
As no colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.

VIABILITY RESULTS
The mean OD value for the test item treated skin samples showed a 96.6% relative viability compared to the negative control.

VALIDITY OF THE TEST
After receipt, the two indicators of the delivered kit were checked in each case. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the two negative control tissues was in the recommended range (0.786).
The two positive control treated tissues showed 0.7% viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 9.4%.
The difference of viability between the two negative control tissue samples in the MTT assay was 8.5%.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Optical Density (OD) and the calculated relative viability % of the samples

Substance	Optical Density (OD)			Viability
		Measured	Blank corrected	(% RV)
Negative Control:	1	0.867	0.820	104.3
Physiological saline	2	0.800	0.753	95.7
(0.9% (w/v) NaCl)	Mean	--	0.786	100.0
Positive Control:	1	0.056	0.009	1.1
Glacial acetic acid:	2	0.050	0.003	0.3
	Mean	--	0.006	0.7
Test Item:	1	0.771	0.724	92.1
PTSM	2	0.842	0.795	101.1
	Mean	--	0.759	96.6

Notes:

1. Mean blank value was 0.047

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

 

HISTORICAL CONTROL DATA

(updated 13 May 2016)

	Negative control (Physiological saline)	Positive control (Glacial acetic acid)
Minimum optical density (OD)	0.611	0.005
Maximum optical density (OD)	1.516	0.051
Mean optical density (OD)	0.871	0.017
Standard Deviation (SD)	0.164	0.010
Number of cases	81	81

Note: All optical density (OD) value measured are background corrected values (measured at 570 ± 30 min)

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with PTSM, the mean cell viability was 96.6% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKIN™(SM) model test with PTSM (Batch number: 609271), the results indicate that the test item is non-corrosive to the skin.

Executive summary:

An in vitro skin corrosivity test of PTSM test item was performed in a reconstructed human epidermis model. EPISKIN™(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

 

Disks of EPISKIN™(SM) (two units) were treated with PTSM test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

 

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

 

Following exposure with PTSM, the mean cell viability was 96.6% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

 

In conclusion, in this in vitro EPISKIN™(SM) model test with PTSM (Batch number: 609271), the results indicate that the test item is non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 May 2017 to 13 May 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:

OECD Guidelines No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (28 July 2015)

Deviations:

yes

Remarks:

Deionised water was used instead of distilled water in the check test (colouring potential of test-item). However, this fact had no impact on the results or integrity of the study.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 761/2009 of 23 July 2009, ANNEX III, B.46., “In Vitro Skin Irritation Reconstructed Human Epidermis Model Test”, amended by Commission Regulation (EU) No 640/2012 of 6 July 2012

Deviations:

yes

Remarks:

Deionised water was used instead of distilled water in the check test (colouring potential of test-item). However, this fact had no impact on the results or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: reconstructed human epidermis model.

Source strain:

other: Batch No.:17-EKIN-019

Details on animal used as source of test system:

EPISKIN TM (SM) (Manufacturer: SkinEthic, France, Batch No.:17-EKIN-019, Expiry Date: 15 May 2017) is a three-dimensional human epidermis model. Adult humanderived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.

Justification for test system used:

The EPISKIN TM (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

Human Skin
EPISKIN TM (SM) (Manufacturer: SkinEthic, France, Batch No.:17-EKIN-019, Expiry Date: 15 May 2017) is a three-dimensional human epidermis model. Adult human derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Quality Control
EPISKIN TM (SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma.
The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKIN TM (SM) test kits used in the present study).

Kit Contents
Units: EPISKIN TM (SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EPISKIN TM (SM) biopsy punch for easy sampling of epidermis
Medium:
A flask of sterile “Maintenance Medium” (Batch No.: 17 MAIN3 018; Exp. Date: 17 May 2017)
A flask of sterile “Assay Medium” (Batch No.: 17 ESSC 018; Exp. Date: 17 May 2017)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

20 μL of test item were applied evenly to the epidermal surface.
50 μL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette.

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (26.2-27.3°C).

Duration of post-treatment incubation (if applicable):

incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere.

Number of replicates:

Three replicates were used for the test item.
Three negative controls and three positive controls were also run in the assay.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item - Mean

Value:

4.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ADDITIONAL CONTROLS
As no colour change was observed after three hours of incubation of the test item in MTT working solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
As the test item was colourless and it had no colouring potential, no additional controls were used for the non-specific OD evaluation.

VIABILITY RESULTS
The OD values for the test item treated skin samples showed 4.2% relative viability.

VALIDITY OF THE TEST
After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the three negative control tissues was in the recommended range (0.716). Standard deviation of the viability results for negative control samples was 1.4.
The positive control treated tissues showed 5.1% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.9.
Note: The OD data of one replicate tissue at the positive control in this study was lower (0.029) than the minimum OD of the historical control range (0.032). This fact has no impact on the results or integrity of the study since the positive control material showed severe effect and was classified as Category 1.
The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 1.4.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Optical Density (OD) and the calculated relative viability % of the samples

Substance	Optical Density (OD)			Viability
		Measured	Blank corrected	(% RV)
Negative Control:	1	0.759	0.713	99.5
Phosphate buffered saline	2	0.755	0.709	99.0
	3	0.774	0.727	101.5
	Mean:	--	0.716	100.0
Positive Control:	1	0.085	0.038	5.3
5% (w/v) SDS solution	2	0.076	0.029	4.1
	3	0.088	0.042	5.8
	Mean	--	0.036	5.1
Test item:	1	0.065	0.019	2.6
PTSM	2	0.084	0.037	5.2
	3	0.081	0.035	4.8
	Mean	--	0.030	4.2

Notes:

1. Mean blank value was 0.047

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

 

HISTORICAL CONTROL DATA

(updated 11 August 2016)

	Negative control (PBS)	Positive control (5% (w/v) SDS solution)
Mean optical density (OD)	0.802	0.094
Standard deviation	0.157	0.048
Minimum optical density (OD)	0.573	0.032
Maximum optical density (OD)	1.362	0.354
Number of cases	111	102

PBS: Phosphate buffered saline

SDS: Sodium dodecyl sulphate

OD: Optical density (absorbance)

Note: All OD values (measured at 570 ± 30 nm) are background corrected values.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In the in vitro EPISKINTM (SM) model test with PTSM (Batch number: 609271), the results indicate that the test item is irritant to skin.

Executive summary:

An in vitro skin irritation test of PTSM test item was performed in a reconstructed human epidermis model. EPISKIN™ (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

 

Disks of EPISKIN™ (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5% CO₂. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5% CO₂protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

 

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

 

Following exposure with PTSM, the mean cell viability was 4.2% compared to the negative control. This is below the threshold of 50%, therefore the test item was considered as being irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

 

In conclusion, in this in vitro EPISKIN™ (SM) model test with PTSM (Batch number: 609271), the results indicate that the test item is irritant to skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

03 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

OECD Guidelines for the Testing of Chemicals 438 (26th July 2013)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

OPPTS 870.2400 (EPA 712-C-98-195) August 1998

Deviations:

no

Principles of method if other than guideline:

Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

Strain of chicken: ROSS 308
Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út 129., Hungary)
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within approximately 2 hours of collection.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 μL of test item was applied.
Negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 μL 5% (w/v) Benzalkonium chloride solution.

Duration of treatment / exposure:

exposure period of 10 seconds.

Duration of post- treatment incubation (in vitro):

The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

3 replicates per group

Details on study design:

Eyes selection
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Preparation of eyes
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

Eyes examination and acclimatization time
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., >0.5) or corneal opacity score (i.e., >0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32 ± 1.5°C) during the acclimatization and treatment periods.

Identification
The eyes were identified by chamber number, marked on the door of the chamber.

The baseline assessments
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye.
The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes.
Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment.
All eyes were considered to be suitable for the assay.

TEST PROCEDURE
Treatment
After the zero reference measurements, the eye (in its retainer) was removed from the chamber, held in a horizontal position and the test item was applied onto the centre of the cornea, taking care not to damage or touch the cornea.
30 μL of test item was applied to the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance.
Negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 μL 5% (w/v) Benzalkonium chloride solution.

Test item removal
The time of application was observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual of the test item if possible.

Observation and assessment of corneal effects
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit BP 900® slit-lamp microscope was used for the measurements.

Morphological effects
Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.

Irritation parameter:

cornea opacity score

Run / experiment:

Test Item - Mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

Test Item - Mean

Value:

0.83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

Test Item - Mean (up to 75 min)

Value:

2.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

Test Item - mean (up to 240 min)

Value:

2.8

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:

The positive control 5% (w/v) Benzalkonium chloride solution was classified as severely irritating, UN GHS Classification: Category 1.
The negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category.

 TEST ITEM

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	2.8%	I
Mean maximum corneal swelling at up to 240 min	2.8%	I
Mean maximum corneal opacity	0.50	I
Mean fluorescein retention	0.83	II
Other observations	None
Overall ICE Class	2xI 1xII

 

POSITIVE CONTROL

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	8.6%	II
Mean maximum corneal swelling at up to 240 min	27.3%	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other observations	Severe loosening of epithelium was observed on three eyes on one eye (1/3) at 75 minutes and on two eyes (2/3) at 240 minutes after the post-treatment rinse.
Overall ICE Class	1xIII 2xIV

 

NEGATIVE CONTROL

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other observations	None
Overall ICE Class	3xI

 

 

Table of individual data PTSM

Study Code:		17/112-038CS														Strain:		ROSS 308
Date of Exposure:		03 May 2017														Test Item:		PTSM
Chamber number ↓	Corneal thickness (instrument units)															Corneal opacity score							Fluorescein retention
Relative observation time (min) →	-45	0	Change	30	Change at 30	75	Change at 75	Max change up to 75	120	Change at 120	180	Change at 180	240	Change at 240	Max change up to 240	0	30	75	120	180	240	Max ∆ Opac	0	30	∆ Flu ret
14	60	60	0.0%	62	3.3%	62	3.3%	3.3%	62	3.3%	62	3.3%	62	3.3%	3.3%	0	0.5	0.5	0.5	0.5	0.5	0.5	0	1	1.0
15	61	61	0.0%	62	1.6%	62	1.6%	1.6%	62	1.6%	62	1.6%	62	1.6%	1.6%	0	0.5	0.5	0.5	0.5	0.5	0.5	0	0.5	0.5
16	60	60	0.0%	62	3.3%	62	3.3%	3.3%	62	6.3%	62	3.3%	62	3.3%	3.3%	0	0.5	0.5	0.5	0.5	0.5	0.5	0	1	1.0
Mean values:					2.8%		2.8%	2.8%		2.8%		2.8%		2.8%	2.8%							0.5			0.83

 

Table of individual data (5 (w/v) % Benzalkonium chloride solution)

Study Code:		17/112-038CS														Strain:		ROSS 308
Date of Exposure:		03 May 2017														Positive Control:		5 (w/v) % Benzalkonium chloride solution
Chamber number ↓	Corneal thickness (instrument units)															Corneal opacity score							Fluorescein retention
Relative observation time (min) →	-45	0	Change	30	Change at 30	75	Change at 75	Max change up to 75	120	Change at 120	180	Change at 180	240	Change at 240	Max change up to 240	0	30	75	120	180	240	Max ∆ Opac	0	30	∆ Flu ret
17	62	62	0.0%	65	4.8%	68	9.7%	9.7%	70	12.9%	74	19.4%	79	27.4%	27.4%	0	4	4	4	4	4	4.0	0	3	3.0
18	62	62	0.0%	66	6.5%	68	9.7%	9.7%	72	16.1%	76	22.6%	79	27.4%	27.4%	0	4	4	4	4	4	4.0	0	3	3.0
19	63	63	0.0%	65	3.2%	67	6.3%	6.3%	70	11.1%	75	19.0%	80	27.0%	27.0%	0	4	4	4	4	4	4.0	0	3	3.0
Mean values:					4.8%		8.6%	8.6%		13.4%		20.3%		27.3%	27.3%							4.00			3.00

 

Table of individual data (physiological saline, 0.9% (w/v) NaCl)

Study Code:		17/112-038CS														Strain:		ROSS 308
Date of Exposure:		03 May 2017														Negative Control:		Physiological saline (NaCl 0.9% w/v)
Chamber number ↓	Corneal thickness (instrument units)															Corneal opacity score							Fluorescein retention
Relative observation time (min) →	-45	0	Change	30	Change at 30	75	Change at 75	Max change up to 75	120	Change at 120	180	Change at 180	240	Change at 240	Max change up to 240	0	30	75	120	180	240	Max ∆ Opac	0	30	∆ Flu ret
20	62	62	0.0%	62	0.0%	62	0.0%	0.0%	62	0.0%	62	0.0%	62	0.0%	0.0%	0	0	0	0	0	0	0.00	0	0	0.00

 

 

TABLES OF ICE CLASSIFICATION

 

ICE classification criteria for corneal thickness:

Mean Corneal Swelling (%)	ICE Class
0 to 5	I
>5 to 12	II
>12 to 18 (>75 min after treatment)	II
>12 to 18 (≤75 mine after treatment)	III
>18 to 26	III
>26 to 32 (>75 min after treatment)	III
>26 to 32 (≤75 min after treatment)	IV
>32	IV

 

ICE classification for corneal opacity:

Mean Maximum Opacity Score	ICE Class
0.0 – 0.5	I
0.6 – 1.5	II
1.6 – 2.5	III
2.6 – 4.0	IV

 

ICE classification for mean fluorescein retention:

Mean Fluorescein Retention Score at 30 minutes post-treatment	ICE Class
0.0 – 0.5	I
0.6 – 1.5	II
1.6 – 2.5	III
2.6 – 3.0	IV

 

Interpretation of results:

GHS criteria not met

Conclusions:

Based on this in vitro eye irritation in the isolated chicken eyes test with PTSM, the test item is non-irritant, UN GHS Classification: No Category.

Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 (26 July 2013).

 

After the zero reference measurements, the eye was held in horizontal position and 30 μL of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 μL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

 

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

 

No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant corneal opacity change (severity 0.5) was noted on three eyes. Slight fluorescein retention change (severity 0.5 or 1) was noted on three eyes. No other corneal effect was observed.

 

SUMMARY TABLE FOR UN GHS CLASSIFICATION

Criteria for “No category” (all true)
3 endpoints classed as I or 2 endpoints class as I and 1 endpoint classed as II:	True
No severe corneal morphological changes	True
Test item was not stuck to the cornea at 240 minutes after the post-treatment rinse	True
Criteria for “Category 1” (one or more true)
2 or more endpoints classed as IV:	False
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes):	False
Corneal opacity = 4 at any time point (in at least 2 eyes):	False
Severe loosening of epithelium (in at least 1 eye):	False
Criteria for “No predication can be made” (one or two true)
Based on the endpoints not classifiable for No Category, or for Category 1:	False
Particles of test item were stuck to the cornea and could not be washed off during the study:	False

 

 

Based on this in vitro eye irritation in the isolated chicken eyes test with PTSM, the test item is non-irritant, UN GHS Classification: No Category.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

In vitro skin corrosion

An in vitro skin corrosivity test of PTSM test item was performed in a reconstructed human epidermis model. EPISKIN™(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

Disks of EPISKIN™(SM) (two units) were treated with PTSM test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Following exposure with PTSM, the mean cell viability was 96.6% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™(SM) model test with PTSM (Batch number: 609271), the results indicate that the test item is non-corrosive to the skin.

In vitro skin irritation

An in vitro skin irritation test of PTSM test item was performed in a reconstructed human epidermis model. EPISKIN™ (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

Disks of EPISKIN™ (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5% CO₂. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5% CO₂protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

 

Following exposure with PTSM, the mean cell viability was 4.2% compared to the negative control. This is below the threshold of 50%, therefore the test item was considered as being irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™ (SM) model test with PTSM (Batch number: 609271), the results indicate that the test item is irritant to skin.

 

Eye irritation - in vitro

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes.

 

After the zero reference measurements, the eye was held in horizontal position and 30 μL of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 μL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

 

No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant corneal opacity change (severity 0.5) was noted on three eyes. Slight fluorescein retention change (severity 0.5 or 1) was noted on three eyes. No other corneal effect was observed.

 

SUMMARY TABLE FOR UN GHS CLASSIFICATION

Criteria for “No category” (all true)
3 endpoints classed as I or 2 endpoints class as I and 1 endpoint classed as II:	True
No severe corneal morphological changes	True
Test item was not stuck to the cornea at 240 minutes after the post-treatment rinse	True
Criteria for “Category 1” (one or more true)
2 or more endpoints classed as IV:	False
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes):	False
Corneal opacity = 4 at any time point (in at least 2 eyes):	False
Severe loosening of epithelium (in at least 1 eye):	False
Criteria for “No predication can be made” (one or two true)
Based on the endpoints not classifiable for No Category, or for Category 1:	False
Particles of test item were stuck to the cornea and could not be washed off during the study:	False

 

Based on this in vitro eye irritation in the isolated chicken eyes test with PTSM, the test item is non-irritant, UN GHS Classification: No Category.

Justification for classification or non-classification

Skin irritation

Based on the in vitro EPISKIN™ (SM) model test with PTSM, the results indicate that the test item is irritant to skin and therefore meets the criteria for classification as Skin Irritant, Category 2.

 

Eye Irritation

Based on the in vitro eye irritation in the isolated chicken eyes test with PTSM, the test item is non-irritant, UN GHS Classification: No Category.

Human Exposure

An incident during use resulted in exposure to a worker that in turn resulted in an adverse response in excess of the response predicted by the in vitro assessments. Following an accidental exposure to an unconfirmed amount of the substance, a worker wearing work wear suffered first and second degree burns. As a precautionary measure, the substance is therefore classified as corrosive.