N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2,5-dimethoxy-4-[(methylamino)sulphonyl]phenyl]azo]naphthalene-2-carboxamide

Endpoint summary

Description of key information

PV32

An in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test according to OECD TG 439. Under the experimental conditions reported, the test item is not irritant to skin.

An in vitro study was performed to assess the corneal irritation and damage potentialof the test item by means of the BCOP assay using fresh bovine corneas (OECD 437: bovine corneal opacity and permeability assay; GLP). Under the experimental conditions reported, the test item does not require classification for eye irritation or eye corrosion.

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:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 439), performed under GLP

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Reconstructed Human Epidermis Test Method (Original Guideline adopted July 22, 2010)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Amount / concentration applied:

Each approximately 10 mg of the neat test item were applied to three EPISKIN (Skinethic) tissues. Additionally, the tissues were wetted with 15 µL of deionised water.

Duration of treatment / exposure:

The skin equivalents were exposed to the test item for 15 minutes. After completion of the treatment the test item was rinsed off and the skin equivalents were incubated for further 42 hours.

Details on study design:

Each approximately 10 mg of the neat test item and 10 µL of the negative control (deionised water) or the positive control (5% Sodium lauryl sulfate, SLS) were applied to each three Episkin (Skinethic) tissues. Additionally, the test item treated tissues were wetted with 15 µl of deionised water. The test item as well as the controls were rinsed off after 15 minutes treatment. After further 42 hours incubation the tissues were treated with the MTT solution for 3 hours following approximately 72 hours extraction of the colorant from the cells. The amount of extracted colorant was determined phototmetrically at a wavelength of 570 nm.

Irritation / corrosion parameter:

other: other: relative absorbance value

Value:

>= 0.867 - <= 1.034

Remarks on result:

other:

Remarks:

Basis: other: three Episkin (Skinethic) tissues. Time point: After treatment with the test item. Remarks: 91.6% (threshold for irritancy: = 50%).

Other effects / acceptance of results:

After treatment with the test item the mean relative absorbance value was reduced to 91.6% (threshold for irritancy: = 50%). Therefore, the test item is not considered to possess an irritant potential.

Results after treatment with test item and 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	Relative Absorbance [%] Tissue 1, 2 + 3**	Standard Deviation [%]	Rel. Absorbance [% of Negative Control]***
Negative Control	15 min	1.106	0.909	1.011	1.009	109.6 90.1 100.2	9.8	100.0
Positive Control	15 min	0.242	0.297	0.193	0.244	24.0 29.5 19.1	5.2	24.2
Test Item	15 min	1.034	0.867	0.872	0.924	102.5 86.0 86.5	9.4	91.6

*       Mean of two replicate wells after blank correction

**      relative absorbance per tissue [rounded values]: (100 x absorbance of tissue) / mean absorbance of negative control
***    relative absorbance per treatment group [rounded values]: (100 x absorbance of test item) / mean absorbance of negative control

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

 

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 91.6% (threshold for irritancy:=50%). Consequently the test item was non irritant to skin.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin.

Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test according to OECD TG 439.

Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes.

Each about 10 mg of the test item were applied to the tissues, wetted with 15 µL of deionised water, and spread to match the tissue size.

10 µL of either the negative control (deionised water) or the positive control (5% Sodium lauryl sulfate) were applied to each tissue.

The test item and the positive and negative controls were washed off the skin tissuesafter 15 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following approximately 72 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD greater or equal 0.6 till less or equal 1.5 for the15 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance compared to the negative control to 24.2%, thus ensuring the validity of the test system.

The standard deviations between the % variabilities of the test item, the positive and negative controls were below 10% (threshold of the "OECD TG 439: 18%), thus ensuring the validity of the study.

After treatment with the test item the mean relative absorbance value was reduced to 91.6% (threshold for irritancy:=50%). Therefore, the test item is not considered to possess an irritant potential.

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:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 437), performed under GLP

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

as of September, 2009

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

GLP compliance:

yes

Amount / concentration applied:

A 20% (w/v) suspension of the test item in physiological saline was prepared. Each 0.75 mL of the suspension were applied to three bovine corneas.

Duration of treatment / exposure:

The corneas were exposed to the test item suspension for 240 minutes.

Details on study design:

After a first opacity measurement of the fresh bovine corneae (t0), 0.75 mL per cornea of a 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item, the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained MEM medium supplemented with sodium bicarbonate and L-glutamine and 1% fetal calf serum (FCS) (complete medium =cMEM). After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t240).
After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically (OD 490) the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

Irritation parameter:

in vitro irritation score

Value:

ca. 2.46

Other effects / acceptance of results:

Relative to the negative control, the test item did not cause any increase of the corneal permeability, and only very slight opacity occurred. The calculated mean in vitro irritation score of 2.46 was below the threshold of = 55.1. Therefore the test item is not corrosive / not severe irritant to the eye according to OECD 437. With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity of the corneae corresponding to a corrosive / severe irritant to the eye.

Results after 240 Minutes Incubation Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		In vitro Score	Mean in vitro irritation score	Proposed in vitro Irritation Scale
		Mean		Mean
Negative Control	1	1.00	0.049	0.057	1.73	1.86	Non corrosive / not severe irritant
	1		0.079		2.19
	1		0.044		1.66
Positive Control	174.00*		0.009*		174.13	183.48	Corrosive / severe irritant
	175.00*		0.028*		175.42
	201.00*		- 0.007*		200.89
Test item	3.00*		0.014*		3.21	2.46	Non corrosive / not severe irritant
	2.00*		- 0.001*		1.98
	2.00*		0.014*		2.21

*corrected values

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item is not corrosive / not severely irritating to the eye.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of the test item by means of the BCOP assay using fresh bovine corneas (OECD 437: bovine corneal opacity and permeability assay; GLP).

Prior to the application, opacity of the fresh bovine corneas was measured (t₀), and 600.91 mg of the test item were suspended in 3.0 mL saline (20% (w/v)) using ultrasonic technique for five minutes. Since the test item could not be suspended homogeneously, each 0.75 mL of the so prepared suspension was distributed to each cornea. Thereby it was taken care of that the corneas were evenly covered with the test item. The positive control was 10% (w/v) Benzalkonium chloride in saline. Saline was used as negative control item.

The exposed corneas were incubated for 240 minutes at 32 +/- 1 °C in complete medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneas and opacity was measured again (t₂₄₀).

After the opacity measurements permeability of the corneas was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 +/- 1 °C.

With the negative control (saline) neither an increase of opacity nor permeability of the corneas could be observed (mean in vitro irritation score 1.86).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity effects (mean in vitro irritation score 183.48) corresponding to a corrosive /severe irritant to the eye.

Relative to the negative control, the test item did not cause any increase of the corneal permeability, and only very slight opacity occurred. The calculated mean in vitro irritation score of 2.46 was below the threshold of >= 55.1. Therefore the test item is not corrosive / not severe irritant to the eye according to OECD 437.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification, as no adverse effects were observed