Benzene, 1,1'-oxybis[methyl-, sulfonated, ammonium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 March - 02 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:

26 July 2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Benzene, 1,1'-oxybis[methyl-, sulfonated, ammonium salts

EC Number:

278-188-0

EC Name:

Benzene, 1,1'-oxybis[methyl-, sulfonated, ammonium salts

Cas Number:

75314-26-0

Molecular formula:

C14H13O4S . NH4 - C70H60O19S6 . 2NH4

IUPAC Name:

Benzene, 1,1'-oxybis[methyl-, sulfonated, ammonium salts

Constituent 2

Reference substance name:

Ammonium sulphate

EC Number:

231-984-1

EC Name:

Ammonium sulphate

Cas Number:

7783-20-2

Molecular formula:

H8N2O4S

IUPAC Name:

Ammonium tetraoxosulfate (VI)

Constituent 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Test material form:

liquid: viscous

Details on test material:

ca 50% aquaous viscous solution
Batch No: INB3209089
Storage: At room temperature, protected from light
Expiring date: 2018-04-30

Specific details on test material used for the study:

Expiration date:10 January 2018

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal
(19.4ºC to 20.3ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4 5 heads/box).
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 fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with 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.
The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

test compound was applied in a single dose (30 µL/eye)

Duration of treatment / exposure:

10sec

Number of animals or in vitro replicates:

Three test item treated eyes, three positive control eyes and one negative control eye were used in this study.

Details on study design:

After the zero reference measurements, one out of three test item treated eyes was held in horizontal position and 30 µL of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) was applied from micropipette onto the centre of the cornea, taking care not to damage or touch the cornea with the application equipment. This procedure was repeated with the remaining two test item treated eyes.
The three positive control eyes were treated with acetic acid 10 % (v/v) solution and one negative control eye was treated with isotonic saline [NaCl (9 g/L saline)] according the above procedure.

Test Item Removal

The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.

Observation and Assessment of Corneal Effects

The control 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 ±5 minutes were considered acceptable.

The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was determined at baseline (t=0) and 30 minutes after the post-treatment rinse.

Retention of Corneas

At the end of the procedures, the corneas were carefully removed from the eyes and placed individually into labeled containers of preservative fluid (4% formaldehyde) for potential histopathology and stored.

Histopathology

After consultation with the Sponsor no histopathology evaluation was performed. Corneas are discarded 2 months after the final report.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Remarks:

Mean %

Run / experiment:

Mean maximum corneal swelling at up to 75 min

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Results 2

Irritation parameter:

in vitro irritation score

Remarks:

Mean %

Run / experiment:

Mean maximum corneal swelling at up to 240 min

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Results 3

Irritation parameter:

cornea opacity score

Remarks:

mean

Run / experiment:

mean maximum

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Results 4

Irritation parameter:

fluorescein retention score

Remarks:

mean

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class III

Results 5

Irritation parameter:

in vitro irritation score

Run / experiment:

1-3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Overall ICE Class:2xI,1xIII

Remarks:

Overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, the test item has been categorized as “No prediction can be made”.

Any other information on results incl. tables

The mean values of the treated eyes for maximum corneal thickness change, corneal opacity, fluorescein retention and other observation (morphological effect etc.) are given below.

 

Test Item: Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%)

 

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	4%	I
Mean maximum corneal swelling at up to 240 min	4%	I
Mean maximum corneal opacity	0.5	I
Mean fluorescein retention	2.0	III
Other Observations	None
Overall ICE Class^1	2xI,1xIII

 

 

Positive Control: Acetic acid 10% (v/v) solution

 

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	21%	III
Mean maximum corneal swelling at up to 240 min	27%	III
Mean maximum corneal opacity	4.0	IV
Mean fluorescein retention	0.3	I
Other Observations	Corneal opacity score 4 was observed in one eye and score 3 was noted in two eyes at 30 minutes after the post-treatment rinse.
Overall ICE Class^1	1xI, 1xIII, 1xIV

 

The positive control Acetic acid 10% (v/v) solution was classed as corrosive/severely irritating,UNGHS Classification: Category 1.

 

Negative Control: NaCl (9 g/L saline)

 

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	5%	I
Mean maximum corneal swelling at up to 240 min	5%	I
Mean maximum corneal opacity	0.5	I
Mean fluorescein retention	0.0	I
Other Observations	None
Overall ICE Class^1	3xI

 

The negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.

 

Applicant's summary and conclusion

Interpretation of results:

other: No ocular corrosion or severe irritation potential was observed. The overall ICE score was 2xI, 1xIII.

Remarks:

According to the guideline OECD 438, the test item has been categorized as “No prediction can be made"

Conclusions:

In this ICET, Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) did not cause ocular corrosion or severe irritation in the enucleated chicken eyes.

Positive and negative controls showed the expected results. The experiment was considered to be valid.


In this in vitro eye irritation study, using the Isolated Chicken Eye model with Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%), no ocular corrosion or severe irritation potential was observed. The overall ICE score was 2xI, 1xIII.

According to the guideline OECD 438, Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, the test item has been categorized as “No prediction can be made”.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose (30 µL/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of chemicals (GHS), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

The test item Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%), positive control (acetic acid 10% (v/v) solution), and negative control (NaCl, 9 g/L saline) were applied in a volume of 30 µL/eye, in such a way to evenly cover the whole cornea surface of each tested eye. Three test item treated eyes, three positive control eyes and one negative control eye were used in this study.

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye.

In this ICET, Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) did not cause ocular corrosion or severe irritation in the enucleated chicken eyes.The overall ICE class was 2xI, 1xIII.

Positive and negative controls showed the expected results. The experiment was considered to be valid.

 

According to the guideline OECD 438, Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) overallin vitroclassification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”.