Sodium bis[3-[[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]-4-hydroxy-N-methylbenzene-1-sulphonamidato(2-)]chromate(1-)

Endpoint summary

Description of key information

The test material was irritaing to rabbits eyes and gave inconclusive results in an in vitro study (BCOP).

In an in vitro reconstructed human skin model the mean relative absorbance value of the test item, corresponding to the cell viability, was 111.7% after taking the correction factor gained in an additional test with a viable tissue into consideration (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin

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:

2016

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UN GHS (2003, last rev. 2015)

Principles of method if other than guideline:

Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Other name: Sodium bis[3-[[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]-4-hydroxy-N-methylbenzene-1-sulphonamidato
(2-)]chromate(1-)
C.I. Index Solvent Orange 41
CAS-No.: 71839-81-1
EINECS / EC-No.: 276-067-7
Appearance: Red powder
Expiry Date: 06.01.2026

Test system:

human skin model

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues

Duration of treatment / exposure:

60 minutes.

Species:

other: reconstituted human epidermis model

Type of coverage:

other: Topical

Preparation of test site:

other: Not applicable

Vehicle:

other: No vehicle used

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
25 mg (~ 39 mg/cm2)

NEGATIVE CONTROL
30 µL DPBS (MatTek) were used as negative control per tissue.

POSITIVE CONTROL
30 µL of a 5% SLS solution in deionized water (MatTek) were used a positive control per tissue.

Duration of treatment / exposure:

60 minutes

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

See table below

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

all

Value:

ca. 111.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The mean relative absorbance value of the test item, corresponding to the cell viability, was 111.7% after taking the correction factor gained in the above mentioned additional test with a viable tissue into consideration (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin. 

Results after treatment with the test item and the controls (exposure interval of 60 minutes):

Dose Group	Tissue No.	Absorbance 570 nm Well 1	Absorbance 570 nm Well 2	Absorbance 570 nm Well 3	Mean Absorbance of 3 wells blank corrected	Mean Absorbance  of 3 wells blank corrected	Mean Absorbance of 3 tissues after blank correction	Rel. Absorb. [%] Tissue 1,2,3
Blank Controls		0.036	0.037	0.036	0.036
Negative Control	1	1.578	1.549	1.558	1.562	1.525	1.811	84.2
	2	2.026	1.984	1.971	1.994	1.957		108.8
	3	2.024	1.971	1.965	1.986	1.950		107.7
Positive Control	1	0.098	0.097	0.097	0.097	0.061	0.078	3.4
	2	0.118	0.121	0.118	0.119	0.082		4.6
	3	0.125	0.126	0.126	0.126	0.089		4.9
Blank Test Item		0.038	0.038	0.037	0.038
Test Item	1	2.095	1.978	1.990	2.021	1.984	2.056	109.5
	2	2.132	2.124	2.132	2.130	2.092		115.5
	3	2.140	2.126	2.127	2.131	2.094		115.6

Dose Group	Tissue No.	Relative Standard Deviation [%]	Mean Relative Absorbance [% of Negative Control] **	Mean Absorbance Blank corrected viable tissue without MTT (Step 2)	Mean Relative Absorbance [% of Negative Control] after colour interference and MTT reduction correction
Blank Controls
Negative Control	1
	2	13.7	100.0
	3
Positive Controls	1
	2	19.0	4.3
	3
Blank Test Item
Test Item	1
	2	3.1	113.6	0.034	111.7
	3

*    relative absorbance per tissue [rounded values]

**   relative absorbance per treatment group [rounded values]

  The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water lead to a change in colour. Therefore an additional experiment for unspecific colour interference was performed and the result was used as correction factor for the evaluation of true tissue viability.

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, was 111.7% after taking the correction factor gained in the above mentioned additional test with a viable tissue into consideration (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin. 

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

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 according to UN GHS and EU CLP regulation.

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.

The test item passed the MTT- pre- test. Because of possible colour interference an additional test with a viable tissue was performed and the result was used as correction factor for data evaluation in the main experiment.

Approximately 25 mg of the test item were applied to each tissue, wetted with 25 µL of DPBS, and spread to match the surface of triplicate tissues.

30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissues each.

The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 43 hours the tissues were treated with the MTT solution for 3 hours following nearly 69 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 OD³0.8 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 4.3% thus ensuring the validity of the test system.

The relative standard deviations between the % variability values of the test item and the negative control in the main test were below 19% (threshold of the "OECD Guideline for the Testing of Chemicals 439:In vitroSkin Irritation: Reconstructed Human Epidermis Test Method”: < 18%). The slight excess of the recommended standard deviation by the positive control (19.0%) is still considered acceptable since the positive control produced a distinct positive result with very low absorbance values, and the mean OD values were within the range of historical control data.

Compared to the relative absorbance value of the negative control the mean relative absorbance value was 111.7% after exposure of the skin tissues to the test item and after taking the correction factor into consideration. This value is above the threshold for irritancy of ≤ 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

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Standard study under GLP, full report, deficient sample characterisation

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Kleintierfarm Madoerin AG CH 4414 Fuellinsdorf/Switzerland
- Age at study initiation: 14 - 15 weeks
- Weight at study initiation: 2.5 - 3.0 kg
- Housing: individual stainless steel cages
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-/+ 3
- Humidity (%): 40 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg / eye
- Concentration (if solution): undiluted

VEHICLE
None

Duration of treatment / exposure:

single exposure; no washing

Observation period (in vivo):

21 days

Number of animals or in vitro replicates:

3

Irritation parameter:

maximum mean total score (MMTS)

Basis:

mean

Time point:

other: 24 h

Score:

3.67

Reversibility:

not fully reversible within: 21 days

Remarks on result:

other: In 1 of 3 animals a slight corneal opacity remaind after 21 days, but there was a clear tendency to recovery

Mean Irritation score for 24 to 72 hours

Animal	Sex	Corneal Opacity	Iris	Redness	Chemosis	Cumul. Score	Mean
40	M	1,00	0,00	1,33	0,67	3,00	2,78
41	M	1,00	0,00	2,00	1,33	4,33
42	F	0,00	0,00	0,67	0,33	1,00

 

Interpretation of results:

irritating

Remarks:

Migrated information

Executive summary:

The test item produced slight but prolonged irritant effects on the rabbit eye.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

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)

Species:

other: Freshly isolated bovine cornea (at least 9 month old donor cattle)

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany

Vehicle:

physiological saline

Controls:

other: 10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl (saline); negative control: Saline

Amount / concentration applied:

Amount(s) applied (volume or weight with unit): 0.75 mL
The test item was tested as a 20% suspension (w/v) in saline.

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

3 corneae per group (test item, negative control, positive control)

Details on study design:

The anterior compartment received the test item suspension or negative or positive control 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.
The incubation time lasted 240 minutes.
Afterwards, the test item or control items, respectively, were rinsed off from the application side with saline, and fresh incubation medium was added into the anterior compartment and opacity was measured (t240).
In the second step of the assay, permeability of the cornea was determined.

SCORING SYSTEM:

Opacity measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.

After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t240).

Permeability Determination
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium will be removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer (Versamax® Molecular Devices). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

DATA EVALUATION:

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.

IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS: In vitro Irritancy Score (according to OECD 437):

≤ 3 No Category (according to GHS)
> 3; ≤ 55 No prediction can be made
> 55 Serious eye damaging according to CLP/EPA/GHS (Cat 1)


Criteria for Determination of a Valid Test

The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), 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:

in vitro irritation score

Run / experiment:

1

Value:

7.34

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

5.95

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

6.27

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

6.52

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritant / corrosive response data:

Relative to the negative control, the test item Savinyl-Orange RLS caused an moderate increase of the corneal opacity and permeability. The calculated mean IVIS was 6.52 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 no prediction for the damage hazard of the test item to the eye can be made.

Results after 240 Minutes Incubation Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposedin vitroIrritancy Score
		Mean		Mean
Negative Control	1	0.33	0.071	0.072	2.07	1.41	Not categorized
	0		0.073		1.10
	0		0.071		1.07
Positive Control	128.67*		-0.015*		128.45	128.75	Category 1
	122.67*		-0.014*		122.46
	135.67*		-0.023*		135.33
Savinyl- Orange RLS	4.67*		0.178*		7.34	6.52	No prediction
	3.67*		0.152*		5.95
	3.67*		0.173*		6.27

*corrected values

Interpretation of results:

study cannot be used for classification

Remarks:

Migrated information

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item is not serious eye damaging (CLP/EPA/GHS (Cat 1) but a prediction for the damage hazard cannot be made (GHS).

Executive summary:

This in vitro study was performed to assess the corneal damage potential of the test item by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t₀), the 20% (w/v) suspension in saline of the test item Savinyl-Orange RLS, the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32± 1 °C. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae andopacity was measured again (t₂₄₀).

After the opacity measurements permeability of the corneae 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 corneae could be observed (mean IVIS = 1.41).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 128.75) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item the test item caused an moderate increase of the corneal opacity and permeability. The calculated mean IVIS was 6.52 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 no prediction for the damage hazard of the test item to the eye can be made.

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 irritant to eyes or skin.

Available studies do not indicate a relevant hazard.