Methyl toluene-4-sulphonate

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl toluene-4-sulphonate

EC Number:

201-283-5

EC Name:

Methyl toluene-4-sulphonate

Cas Number:

80-48-8

Molecular formula:

C8H10O3S

IUPAC Name:

methyl 4-methylbenzene-1-sulfonate

Test material form:

solid

Details on test material:

Name: PTSM
Chemical name: Methyl toluene-4-sulphonate
CAS number: 80-48-8
Batch/Lot Number: 609271
Description: White to pale yellow solid
Expiry Date: 01 March 2018
Purity: 99.7%
Storage Conditions: Room temperature
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Specific details on test material used for the study:

No further details specified in the study report.

Test animals / tissue source

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.

Test system

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.

Results and discussion

In vitro

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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.

Any other information on results incl. tables

 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

 

Applicant's summary and conclusion

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.