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

Endpoint summary

Description of key information

Skin irritation

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) is considered to be non-irritant to skin and is therefore not classified (not classified with skin irritation according to the CLP regulation 1272/2008/EC).

Eye irritation

Study in progress (05/2018). Based on available data (supporting studies) it is assumed that the substance is not irritating to the eyes (not classified with eye irritation according to the CLP regulation 1272/2008/EC).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The study may be used to draw a conclusion on the non corrosive/ non-irritating properties to skin of the tested substance in combination with other supporting documentation. The report does not include details on the study method or the compostion of the test substance.

Qualifier:

no guideline followed

Species:

rabbit

Type of coverage:

not specified

Preparation of test site:

not specified

Vehicle:

not specified

Amount / concentration applied:

Not specified

Duration of treatment / exposure:

Not specified

Observation period:

3 days

Number of animals:

5

Irritation parameter:

erythema score

Basis:

animal: #1,2,3,4 and 5

Time point:

other: 24 and 72 h

Score:

1

Max. score:

1

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

edema score

Basis:

animal: # 1 and #2

Time point:

other: 24 and 72 h

Score:

1

Max. score:

1

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

edema score

Basis:

animal #3

Time point:

other: 24 and 72 h

Score:

0.5

Max. score:

1

Reversibility:

fully reversible within: 72 h

Irritation parameter:

edema score

Basis:

animal #4

Time point:

other: 24 and 72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 72 h

Irritation parameter:

edema score

Basis:

animal #5

Time point:

other: 24 and 72 h

Score:

0

Max. score:

0

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not irritant to the skin

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 March, 2017 - 17 April, 2018

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:

28 April 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.

Version / remarks:

February 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the
EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT
reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the
purposes of distinguishing between skin irritating and
no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection;
Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France, 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.

Supplier: SKINETHIC Laboratories
4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
Batch No.: 18-EKIN-010
Expiry date: 12 March 2018

EpiSkinTMSM 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
an MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS).

EpiSkinTMSM KIT Contents

Units: EpiSkinTMSM 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: EpiSkinTMSM biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium” for incubations.
(Batch No.: 18 MAIN3 011; Exp. Date: 14 March 2018)
A flask of sterile “Assay Medium” for use in MTT assays.
(Batch No.: 18 ESSC 009; Exp. Date: 14 March 2018)

The EpiSkinTMSM units were kept in their packaging at room temperature until the pre-incubation was started. The maintenance and assay medium were stored at 2-8°C.

Indicator for potential false viability

Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when
the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material acts directly on MTT (MTT-reducer),
is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test substance interference with the viability measurement.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

10 µL test item was applied evenly to the epidermal surface of each of the three test skin units.

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1h)

Number of replicates:

Three replicates were used for the test item and controls, respectively.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

92

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

122

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

116

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

1-3

Value:

110

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other: mean relative viability%

Run / experiment:

1-3

Value:

82

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:

Validity of the Test

The mean OD value of the three negative control tissues was 0.911. The mean OD value obtained for the positive control was 0.062 and this result
corresponds to 7 % viability when compared to the results obtained from the negative control. Each calculated standard deviation value (SD) for the %
viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Indicator for potential false viability

Possible direct MTT reduction with test item:
No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data
calculations were not necessary. A false estimation of viability can be precluded.

Colouring potential of test item:
As the test item has an intrinsic colour (brown), two additional test item-treated tissues were used for the non-specific OD evaluation. The mean OD
(measured at 570 nm) of these tissues was determined as 0.257. The Non Specific Colour % (NSC %) was calculated as 28.2%. Therefore additional data
calculation was necessary.

Cell viability

The results of the optical density (OD) measured at 570 nm of each replicate .

OD values and viability percentages of the controls and test item:

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1x PBS	1	0.898	99
	2	0.920	101
	3	0.915	100
	mean	0.911	100
	standard deviation (SD)		1.28
Positive Control: SDS (5 % aq.)	1	0.066	7
	2	0.043	5
	3	0.077	8
	mean	0.062	7
	standard deviation (SD)		1.91

 

OD values and viability percentages of the test item (including corrected values):

Test Item	Optical Density (OD)		TODTT	Viability (%)	Relative Viability (%)
Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%)	1	0.842	0.585	92	64
	2	1.107	0.850	122	93
	3	1.056	0.799	116	88
	mean	1.002	0.745	110	82
	standard deviation (SD)			15.43	15.43

OD values and NSC % of additional control:

Additional colour control	Optical Density (OD)		Non Specific Colour %(NSC %)
Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) (test item treated tissueswithout MTT incubation)	1	0.179	28.2
	2	0.335
	mean	0.257

Remark:TOD_TT:true MTT metabolic conversion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Executive summary:

EpiSkin^TMSM test of Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439,28 July 2015.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes
(± 0.5 min)at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1°C for 42 hours (±1 hour) in an incubator with 5±1% CO₂. The viability of each disk was assessed by incubating the tissues for 3 hours (±5 min) with MTT solution at 37±1°C in 5±1% CO₂protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test item has an intrinsic colour (brown), therefore two additional test item treated tissues were used for the non-specific OD evaluation.

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (=) to 50%

of the negative control.

In thisin vitroskin irritation test using the EPISKIN model, the test item Benzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%) did not show significantly reduced cell viability in comparison to the negative control

(mean relative value: 82 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid.

 

The results obtained from thisin vitroskin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test itemBenzene, 1,1’-oxybis(methyl-, sulfonated, ammonium salt (ca 50%)is considered to be non-irritant to skin and is therefore not classified(UN GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The study may be used to draw a conclusion on the non corrosive/ non-irritating properties to the eyes of the tested substance in combination with other supporting documentation. The report does not include details on the study method or the compostion of the test substance

Qualifier:

no guideline followed

GLP compliance:

not specified

Species:

rabbit

Strain:

not specified

Vehicle:

not specified

Controls:

not specified

Observation period (in vivo):

3 Days

Number of animals or in vitro replicates:

6

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 h

Irritation parameter:

cornea opacity score

Basis:

animal #5

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

cornea opacity score

Basis:

animal: #1, #2, #3 and #6

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal: #1,2,3,4,5 and 6

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

chemosis score

Basis:

animal: #4 and #5

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

animal: #1, 2,3 and 6

Time point:

24/48/72 h

Score:

0.66

Max. score:

1

Reversibility:

fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal: #1 and #3

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal: #2 and #6

Time point:

24/48/72 h

Score:

1.66

Max. score:

2

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal: #4 and #5

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not classified as eye irritant.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The study may be used to draw a conclusion on the non corrosive/ non-irritating properties to the eyes of the tested substance in combination with other supporting documentation. The report does not include details on the study method or the compostion of the test substance.

Qualifier:

no guideline followed

Principles of method if other than guideline:

9 white Rabbitswere instilled once daily during 5 consecutive days one mililter of a 1% dilution or the test material on the left eye. On 3 animals the eyes were not rinsed. On 3 animal the eyes were rinsed after 3 seconds with 20 ml water. The eyes of the remainig three animals were rinsed after 4 seconds with 20 ml water.

GLP compliance:

no

Species:

rabbit

Strain:

not specified

Remarks:

white

Vehicle:

not specified

Controls:

not specified

Amount / concentration applied:

1 ml of solution with 1% of test item

Duration of treatment / exposure:

5 consecutive days treatment
on 3 rabbits the eye was rinsed after 2 seconds exposure, on 3 animal after 4 seconds exposure and od the rest 3 animal the eye was not rinsed at all.

Observation period (in vivo):

Not specified

Number of animals or in vitro replicates:

3

Details on study design:

9 white Rabbitswere instilled once daily during 5 consecutive days one mililter of a 1% dilution or the test material on the left eye. On 3 animals the eyes were not rinsed. On 3 animal the eyes were rinsed after 3 seconds with 20 ml water. The eyes of the remainig three animals were rinsed after 4 seconds with 20 ml water.

Irritation parameter:

other:

Basis:

animal: 1-9

Reversibility:

not specified

Remarks on result:

no indication of irritation

Remarks:

No indication of irritation like redness, swelling or secretion were observed

Interpretation of results:

GHS criteria not met

Conclusions:

No indication of eye iritation was observed.

Reference 3

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

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

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)

Specific details on test material used for the study:

Expiration date:10 January 2018

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.

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.

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

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

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

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

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”.

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.

 

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”.

Reference 4

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018 - study in progress

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)

GLP compliance:

yes (incl. QA statement)

Irritation parameter:

in vitro irritation score

Value:

> 0

Remarks on result:

other: preliminary results, study in progress

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available