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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: according to OECD 439 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: UN GHS (published 2003, last (3rd) revision 2009

Deviations:

no

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)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

275-863-1

EC Name:

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

Cas Number:

71701-14-9

Molecular formula:

C34H28Cl2CoN10O8S2.Na

IUPAC Name:

Cobaltate(1-), bis[3-[2-[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-(oxo-kO)-1H-pyrazol-4-yl]diazenyl-kN1]-4-(hydroxy-kO)-N-methylbenzenesulfonamidato(2-)]-, sodium (1:1)

Test animals

Species:

other: reconstituted human epidermis model

Strain:

other: reconstituted human epidermis model

Test system

Type of coverage:

other: Topical

Preparation of test site:

other: Not applicable

Vehicle:

other: No vehicle used

Amount / concentration applied:

Test Item Preparation:
The test material was crushed and ground in a mortar with pestle to improve the consistency. Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues, wetted with 25 µL DPBS, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.

Negative Control:
30 µL DPBS (MatTek) was used as negative control per tissue.

Positive Control:
30 µL of a 5% SLS solution in deionised water (MatTek) was used a positive control per tissue, freshly prepared prior to the start of the experiment.


Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes.

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 tissue.
30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissue 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 69.75 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

Duration of treatment / exposure:

60 minutes

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

Test for Direct MTT Reduction and Colour Interference
For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. For this purpose, approximately 25 mg of the test item were added to 1 mL of MTT solution (MTT (Sigma, Germany) concentrate diluted with MTT diluent (DMEM, Gibco, Germany) (resulting: 1 mg/mL)). This mixture was incubated in the dark at 37 ± 1.5 °C (5 ± 0.5% CO2) for 60 minutes.
Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer.
Prior to the start of the test, the test item’s colour interference potential had to be evaluated. For this purpose approximately 25 mg of the test item were mixed with 300 µL of deionised water. This mixture was incubated for 60 minutes at 37 ± 1.5 °C (5 ± 0.5% CO2).
Since the colour of the test item / water mixture was intensive, an additional functional check was performed. The coloured test item was applied to one viable tissue, which underwent the entire skin irritation test (exposure period: 60 minutes; recovery period: about 41 hours; extraction period: 70 hours), but was incubated with medium instead of MTT solution during the MTT incubation step.
Since the OD of the tissue treated by the coloured test item was > 30% (651.8%) of the DPBS treated control tissue, the test item could have been considered as incompatible with the test. But according to an expert judgment test item particles could have been trapped in the tissue and could not be removed in the washing step. The gained high absorption value was most probably caused by reflection of the remaining particles.
Therefore, the real MTT OD (unaffected by interference with the coloured test items) was calculated using following formula:
OD = ODcoloured tissue (MTT assay) – ODcoloured tissue (no MTT assay)

Performance
Pre-warming of EpiDerm™ Tissues
One day prior to the performance, the plastic bag containing the 24-well plate with epidermal tissues was opened under sterile conditions. Under an airflow using forceps, the gauze was removed and the inserts were taken out. Any remaining agarose that adheres to the outer sides of the inserts was removed by gentle blotting on the sterile filter paper or gauze, and the tissues were placed in the empty, sterile 6-well plate. Prior to the exposure of the test item and of the controls the EpiDerm™ tissues were inspected for quality: If necessary, it was taken care, that
1. air bubbles between agarose and insert were not > 30% of the total surface,
2. liquid on top of the insert was removed with steriles cotton tips,
3. if again moisture is observed on top of the inserts after the pre-incubation or in
case of visible defects the respective skin models were discarded.
0.9 mL of the assay medium (20 – 25 °C) was pipetted into each well of sterile 6-well plates. The inserts with the EpiDerm™ tissues were placed in the upper wells, and were pre-incubated for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH). Following, the inserts were transferred from upper wells into the lower wells of the 6-well plates, and, the pre-incubation was continued for further 22 hours and 20 minutes (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).
Treatment
After pre-incubation of EpiDerm™ tissues was completed, medium was replaced by 0.9 mL of fresh medium per well. The negative and positive control, the vehicle control, and the test item were added into the insert atop the corresponding EpiDerm™ triplicate tissues. The treatment time was 60 minutes in total. Within this period the 6-well plates were put into 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 immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were carefully dried using sterile cotton tipped swap. The tissues were then transferred into new 6-well plates with 0.9 mL of fresh assay medium in the upper row. The inserts were placed in the prepared holding plate. Tissues were incubated for 23 hours and 38 minutes 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 19 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was 42 hours and 30 minutes.

MTT Assay
On the day of testing the MTT concentrate was diluted with the MTT diluent (1 mg/mL). The 24-well plates were prepared before the end of the tissue pre-warming period. A volume of 300 µL of the MTT solution was added to each well and the plates were kept in an incubator (37 ± 1 °C, 5 ± 0.5 % CO2) until further use.
After the 42-hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT-plates. 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 three times with DPBS. Inserts were transferred onto new 24-well plates. The inserts were immersed into extractant solution by gently pipetting 2 mL extractant solution (isopropanol) in each insert. The level rose above the upper edge of the insert, thus tissues were completely covered from both sides. The 24-well plate was sealed to inhibit the isopropanol evaporation.
The formazan salt was extracted for 70 hours without shaking in the refrigerator.
After the extraction period was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The 24-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. 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.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

>= 84.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

threshold for irritancy:≤50%

In vivo

Irritant / corrosive response data:

Example:
Compared to the relative absorbance value of the negative control the mean relative absorbance value was reduced to 84.5% (corrected value) 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.

Other effects:

No

Any other information on results incl. tables

Results after treatment with the test item, and the controls

Dose Group	Treat-ment Interval	Absor-bance 570 nm Tissue 1*	Absor-bance 570 nm Tissue 2*			Absor-bance 570 nm Tissue 3*		Mean Absor-bance of 3 Tissues			Rel. Absor-bance [%] Tissue 1, 2 + 3**		Relative Standard Deviation [%]	Mean Rel. Absorbance [% of Negative Control]***
Negative Control	60 min	1.869	1.894			1.949		1.904			98.2 99.5 102.3		2.1	100.0
Positive Control	60 min	0.102	0.105			0.074		0.094			5.4 5.5 3.9		18.0	4.9
Test Item	60 min	1.578	1.582			1.713		1.624			82.9 83.1 90.0		4.7	85.3
Colour interference pre-experiment: Functional Test without MTT
		Absorbance 570 nm Well 1			Absorbance 570 nm Well 2		Absorbance 570 nm Well 3			Mean Absorbance of 3 Wells		Mean Absorbance subtracted by blank		Mean Rel. Absorbance [% of Negative Control]***
Negative Control	60 min	0.0381			0.04		0.0398			0.0393		0.0028		100.0
Test Item without MTT	60 min	0.0617			0.0527		0.0504			0.0549		0.0185		651.8
Data correction procedure
	Mean Absorbance of 3 Tissues (main experiment)			Mean Absorbance of 3 Wells (Functional Test without MTT)					ODMTT– ODno MTT			Corrected Mean Rel. Absorbance [% of Negative Control]***
Negative Control	1.904			0.0028					1.901			100.0
Test Item	1.624			0.0185					1.606			84.5

* Mean of three replicate wells after blank correction

**  Relative absorbance per tissue [rounded valuea]: 100 x absorbance_^tissue

(mean absorbance _{negative control})

*** Relative absorbance per treatment group [rounded values]:  100 x (mean absorbance _{test item/positive control})

(mean absorbance _{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. Due to the intrinsic colour of the test item, an additional test with viable tissues (entire test procedure) but without MTT was performed. A correction from the mean relative absorbance value of the test item, corresponding to the cell viability was necessary.

After correction of absorbance (OD = absorbance_MTT– absorbance_{no MTT}) the mean relative absorbance of Savinyl-Gelb 2RLS, trocken, corresponding to the cell viability was 84.5% (threshold for irritancy:≤50%), consequently the test item was not irritant to skin.

 

 

 

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

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 by means of the Human Skin Model Test.

The test item passed the MTT- pre-test. Due to its intrinsic colour, an additional test with one viable tissue (entire test procedure), but with medium instead of MTT addition was performed.

Since the OD of the tissue treated by the coloured test item was > 30% (651.8%) of the DPBS treated control tissue in this test,the test item could have been considered as incompatible with the test. But according to anexpert judgment test item particles were most probably trapped in the tissue and could not be removed in the washing step. The gained high absorption value was most probably caused by reflection of the remaining particles.

Therefore, the real MTT OD (unaffected by interference with the coloured test items) was calculated by subtracting the OD_{no MTT}from the OD_{with MTT.}

The test item, the negative control (DPBS), and the positive control (5% SLS) were applied to each of triplicate tissue.

The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 41 hours and 50 minutes the tissues were treated with the MTT solution for 3 hours following 70 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 at or above 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 4.9% thus ensuring the validity of the test system.

The relative standard deviations between the % variabilities of the test item, the positive and negative controls in the main test were 18% (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.

Compared to the relative absorbance value of the negative control the corrected mean relative absorbance value was reduced to 84.5% (corrected value) 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.