5,5'-sulfonylbis(2-benzofuran-1,3-dione)

Endpoint summary

Description of key information

Skin irritation: not irritating (OECD 439; GLP compliant)
Eye irritation: a prediction if the test item is irritating to the eyes or not cannot be made. However, the test item can be considered as not damaging to the eye (OECD 437; GLP compliant)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-11-11 to 2014-11-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2013-07-26

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) for use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200), Rev. 1/19/2010.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2013-04-11

Details on test animals or test system and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals.

Vehicle:

other: Dulbecco's Phosphate Buffered Saline

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):approximately 25 to 26.5 (~ 43 mg/cm²) of the tets item were applied to the tissues wetted with 25 µL vehicle

Duration of treatment / exposure:

60 minutes

Observation period:

not applicable

Number of animals:

not applicable

Details on study design:

CELL CULTURE:
- Epi-200 SIT kits (Lot no.: 19695, Kit B) and MTT-100 assays diluent were purchased from MatTek Corporation (82105 Bratislava, Slovakia).
- EpiDerm™ tissue (surface 0.6 cm²) consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

TEST FOR DIRECT MTT REDUCTION COLOUR INTERFERENCE
1) Colour interference:
- prior to the treatment the colour interference potential of the the test item was evaluated by spectral analysis of the test item mixed with water.
- ~25 mg of the test item was mixed with 300 µL of deionised water.
- test item suspension was incubated for 60 minutes at 37 ± 1.5°C (5 ± 0.5% CO2)
2) Direct MTT Reduction:
- approximately ~25 mg of the test item were added to 1 mL of MTT solution (MTT diluted with DMEM (resulting: 1 mg/mL)) and the mixture was incubated in the dark at 37 ± 1.5°C (5 ± 0.5% CO2) for 60 minutes.
- untreated MTT medium was used as control.
- if the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.

TREATMENT:
- one day prior to the performance, the tissues were placed in the sterile 6-well plate.
- prior to the exposure of the test item and of the controls the EpiDerm™ tissues were inspected for quality.
- 0.9 mL of the assay medium (20 - 25°C) was pipetted into each well of sterile 6-well plates and the inserts with the EpiDerm™ tissues were pre-incubated for a total duration of about 24.5 hours (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).
- after the pre-incubation of EpiDerm™ tissues, medium was replaced by 0.9 mL of fresh medium per well in the 6-well plates.
- the negative control (Dulbecco's Phosphate Buffered Saline (DPBS) (30 μL)) and positive control (5% sodium lauryl sulphate (SLS) solution in deionised water (30 μL)) and the test item were added into the insert atop the corresponding EpiDerm™ triplicate tissues.
- treatment time was 60 minutes in total. Within this period the 6-well plates were placed in the incubator for 35 minutes at 37 ± 1.5°C, 5 ± 0.5% CO2. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.
- after the end of the treatment interval the inserts were removed from the plate and tissues were rinsed with DPBS in order to remove any residual test material.
- tissues were transferred into new 6-well plates with 0.9 mL of fresh assay medium and tissues were incubated for approximately 23 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2.
- after incubation the inserts were transferred into new 6-wells plates containing fresh medium. Thereafter tissues were incubated for another 18.5 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2.
- complete incubation time was about 42 hours.

CELL VIABILITY TEST:
- cell viability is measured by dehydrogenase conversion of MTT, in cell mitochondria, into a blue formazan salt.
- after the 42-hours incubation period, culture inserts were transferred from the holding plates to plates containing 300 µL of MTT solution.
- after a 3-hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2), the MTT solution was aspirated from the wells, and the wells were rinsed with DPBS. Inserts were transferred onto new 24-well plates.
- the inserts were immersed into extractant solution (isopropanol).
- the formazan salt was extracted for about 69 hours at room temperature.
- after the extraction period, the inserts were pierced to allow the extract to run into the well from which the insert was taken.
- well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
- per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure.
- optical density (OD) was read in a microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1) with a 570 ± 1 nm filter.
- mean values were calculated from the 3 wells per tissue.

EVALUATION OF RESULTS:
- mean OD of the three negative control tissues was calculated.
- for each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula: Relative viability (%) = [(meanOD test item/positive control)/ ODmean of negative control] x 100
- for the test item and the positive control the mean relative viability ± rel. standard deviation of the three individual tissues was calculated and used for classification.
- an irritation potential of a test item according to EU classification R38 (according to directive 67/548/EEC), H315 (according to regulation (EC) 1272/2008) is recommended if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control.

Irritation / corrosion parameter:

other: other: relative viability(%)

Value:

88.9

Remarks on result:

other:

Remarks:

Basis: mean. Time point: after 60 minutes incubation. Reversibility: no data. (migrated information)

Irritant / corrosive response data:

Compared to the relative absorbance value of the negative control the mean relative absorbance value was reduced to 88.9% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

HISTORICAL DATA

Positive control		Negative control [OD570]
Mean viability	5.6%	Mean absorption	1.796
Rel. standard deviation	2.4%	Rel. standard deviation	0.281
Range of viabilities	2.9 – 11.3%	Range of viabilities	1.397 – 2.651
Mean absorption	0.098
Rel. standard deviation	0.038
Range of absorbance	0.030 - 0.194

Data of 125 studies performed from November 2011 until May 2014.

Results after treatment with the test item and the controls

Dose Group	Treatment Interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Mean Rel. Absorbance [% of Negative Control]**
Negative control	60 minutes	2.110	2.126	2.147	2.128	100.0
Positive control	60 minutes	0.098	0.108	0.087	0.098	4.6
Test item	60 minutes	1.899	1.811	1.962	1.890	88.9

* Mean of three replicate wells after blank correction

** Relative absorbance per treatment group [rounded values]: [100 X (mean absorbance test item / positive control)] / (mean absorbance negative control)

- the optical pre-experiment (colour interference pre-experiment) to investigate the test item's colour change potential in water did not led to a change in colour. The OD570 of the test item suspension in water was 0.185.This value was too low to have an influence on the outcome of the study. Consequently, an additional control with viable tissues without MTT application was not necessary to be performed.

- the optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour. Conseqeuntly, an additional control with freeze-killed tissues was not necessary to be performed.

- after treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.

- treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.6% thus ensuring the validity of the test system.

- the rel. standard deviations between the % variabilities of the test item, the positive and negative controls were below 11% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item is not an irritant to the skin.
According to the Directive 67/548/EEC and its subsequent amendments, the test substance is not irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not classified as irritating to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

2015-01-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted 2013-07-26

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2013-04-11

Details on test animals or tissues and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals.

Vehicle:

physiological saline

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL of the test item solution
- Concentration: test item was tested as a 20% suspension (w/v) in saline (0.9% (w/v)

Duration of treatment / exposure:

240 minutes

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

not applicable

Details on study design:

COLLECTION OF BOVINE EYES
- isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir and were transported in Hank's buffered salt solution (HBSS) supplemented with streptomycin / penicillin at ambient temperature.

PREPARATION OF CORNEAE
- all eyes were carefully examined macroscopically for defects.
- the cornea was carefully removed from the eye.
- each cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. Both compartments of the holder were filled with incubation medium.
- for equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
- at the end of the incubation period, the basal opacity was determined (t0) of all cornea.
- each corneae with a value of the basal opacity > 7 was discarded.

EXPOSURE OF THE CORNEAE TO THE TEST GROUPS
- the anterior compartment received the test item suspension or negative control (saline) or positive control (10% (w/v) benzalkonium chloride in 0.9% (w/v) NaCl (saline)) at a volume of 0.75 mL each on the surface of the corneae.
- the corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath (incubation time: 240 minutes).
- the test item, positive control and negatice control were tested in triplicate.
- the test item or control items were rinsed off with saline
- incubation medium was added into the anterior compartment and opacity was measured (t240)
- permeability of the corneae was determined.

PERMEABILITY MEASUREMENT
- after the final opacity measurement, the incubation medium was removed from the anterior compartment and replaced by 0.5% (w/v) sodium fluorescein solution in HBSS.
- corneae were incubated in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C.
- incubation medium from the posterior compartment was removed, well mixed and the optical density at 490 nm was determined with a spectrophotometer. The absorbance values were determined using the software SoftMax Pro Enterprise, version 4.7.1.

EVALUATION OF RESULTS
- Opacity: the change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- Permeability: the corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IN VITRO IRRITATION SCORE CALCULATION
The following formula was used to determine the in vitro irritation score (IVIS) of the negative control:
In vitro Irritation Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro irritation score of the positive control and the test item:
In vitro Irritation Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group was calculated from the IVIS values.
Depending on the score obtained, the test item was classified into the following category according to OECD guideline 437 (table 1 in the field "Any other information on materials and methods incl. tables" below).

CRITERIA FOR DETERMINATION OF A VALID TEST
The test was acceptable if
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean, and if
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Irritation parameter:

other: in vitro irritancy score

Basis:

mean

Time point:

other: 240 minutes

Score:

8.29

Irritant / corrosive response data:

Relative to the negative control, the test item 4,4’-sulfonyldiphthalic acid dianhydride caused an increase of the corneal opacity but not of the permeability. The calculated mean in vitro irritancy score IVIS was 8.29 (threshold for serious eye damage: IVIS ≥ 55). Hence, classification as damaging to eyes Cat.1 is not required (irritation score < 55), but no prediction on eye irritation or non-eye irritation can be made, since the irritancy score is > 3. This is in accordance with OECD 437.

Table 1: Results after 240 minutes incubation time

Test group	Opacity value = Difference (t240 – t0) of opacity		Permeability at 490 nm (OD490)		In vitro irritancy score	Mean in vitro irritancy score
		Mean		Mean
Negative control	0	0	0.052	0.051	0.78	0.77
	0		0.051		0.77
	0		0.050		0.75
Positive control	205.00*		0.240*		208.60	201.62
	188.00*		0.245*		191.68
	202.00*		0.173*		204.60
4,4‘ –sulfonyldiphthalic acid dianhydride	4.00*		-0.005*		3.93	8.29
	8.00*		-0.002*		7.97
	13.00*		-0.002*		12.97

* corrected values

- With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.77).

- The positive control ( 10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS =201.62).

Table 2: Historical data

	Positive control	Negative control
Mean IVIS	176.58	1.71
Standard deviation	41.85	0.76
Range of IVIS	62.60 - 292.29	0.00 – 3.00

Values of 206 studies with solid test items performed from February 2007 until December 2014

Interpretation of results:

other: a prediction if the test item is irritating to the eyes or not cannot be made. However, the test item can be considered as not damaging to the eye.

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, for 4,4’-sulfonyldiphthalic acid dianhydride a prediction if the test item is irritating to the eyes or not cannot be made. However, the test item can be considered as not damaging to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation:

An in vitro skin irritation study according to OECD 439 (k_Roth_2015) is considered to be reliable without restrictions and the results indicate that the test item is not irritating to skin.

Eye irritation:

An in vitro eye irritation study according to OECD 437 (k_Roth_2015) is considered to be reliable without restrictions. A prediction if the test item is irritating to the eyes or not cannot be made. However, the test item can be considered as not damaging to the eye.

Justification for selection of skin irritation / corrosion endpoint:
GLP guideline study conducted with the test item

Justification for selection of eye irritation endpoint:
GLP guideline study conducted with the test item

Justification for classification or non-classification

Skin irritation:

The test item does not possess a skin irritation potential and does not require classification as skin irritant according to Directive 67/548/EEC and its subsequent amendments and Regulation (EC) No 1272/2008 and its subsequent regulations.

Eye irritation:

The test item 4,4’-sulfonyldiphthalic acid dianhydride (DSDA) caused an increase of the corneal opacity but not of the permeability (BCOP - OECD 437). The calculated mean in vitro irritancy score IVIS was 8.29 (threshold for serious eye damage: IVIS ≥ 55). Hence, classification as damaging to eyes Cat.1 is not required (irritation score < 55), but no prediction on eye irritation or non-eye irritation can be made, since the irritancy score is > 3.

In conclusion, the substance can be considered as being not damaging to eyes and is classified by worst case as being irritating to the eye (H319) in accordance with Regulation (EC) 1272/2008.