Chromium (III) complexes with 2-[{2,4-dihydroxy-3-[(5-sulfo-1-naphthyl)diazenyl]phenyl}diazenyl]benzoic acid (1:2), sodium salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 2015

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Chromium (III) complexes with 2-[{2,4-dihydroxy-3-[(5-sulfo-1-naphthyl)diazenyl]phenyl}diazenyl]benzoic acid (1:2), sodium salts

EC Number:

700-953-9

Molecular formula:

C46H26CrN8O14S2.3Na

IUPAC Name:

Chromium (III) complexes with 2-[{2,4-dihydroxy-3-[(5-sulfo-1-naphthyl)diazenyl]phenyl}diazenyl]benzoic acid (1:2), sodium salts

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Details on test animals or test system and environmental conditions:

test in vitro

Test system

Amount / concentration applied:

20 mg directly applied

Duration of treatment / exposure:

3, 60 and 240 minutes

Details on study design:

Experimental Design

Preliminary Test

Indicator for potential false viability
Optical properties of the test item 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 item 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 item interference with the viability measurement.(i.e. use of killed epidermis).
The test item “Acid Brown 432”, interfered with MTT (MTT-reducer), it was naturally coloured and it coloured the skin during the treatment.


Check-method for possible direct MTT reduction with test item
20.2 mg of “Acid Brown 432”, were added directly to 2.0 mL of MTT 0.3 mg/mL solution and mixed. The mixture was incubated for three hours (± 5 min) at 37°C, 5% CO2, 95% humidified atmosphere, protected from light. After the incubation period, a strong interaction with MTT was observed. The MTT solution, starting from yellow colour, changed becoming brown, so the test item interacted with MTT solution.
In this case, the use of killed epidermis was necessary, because the test item affected the OD with a-specific MTT reduction, so that there was the possibility of an overvalue of the OD.


Check-method to detect the colouring potential of test item
In order to evaluate the test item intrinsic colour or its ability to become coloured in contact with water (simulating a tissue humid environment), 10.2 mg of “Acid Brown 432” were added to 90 µL of deionized water in a transparent micro-tube and mixed for 15 minutes.
At the end of the shaking period, the operator checked the colour by an unaided eye assessment, and also in this case, a strong brown colour was detected. Therefore, since the test item showed ability to become coloured in contact with water, additional treated tissues were necessary for the non specific OD evaluation.


Test

Pre-incubation and Application (Day 0-1)

The Maintenance Medium was pre-warmed to 37°C. The appropriate number of assay plate wells were filled with the pre-warmed medium (2.0 mL/well). A total number of 20 vital skin inserts were used: two skin units were used for the test item and two were used for the Negative Control (NC) for each of the three different test exposure times (= 6 units for test item + 6 units for NC) and only two skin units were used for the Positive Control (PC) (mandatory only at the 240 min. exposure time). ). Two wells were included for every incubation time (total number 6), in order to quantify the NSC.
Moreover, for every incubation time, two units with killed cells + two units with killed cells for the Negative Control at 240 minutes application time (total number 8) were de-frozen in maintenance medium about half an hour before use.

The epidermis units were placed above the medium, avoiding bubbles between the skin and the maintenance, each one into its prepared well and then incubated for about 24 hours (provided time between 1 e 48 hours) at 37°C, 5% CO2, 95% humidified atmosphere.

After the pre-incubation period, the skin units were transferred to other plates filled with 2.0 mL/well of assay medium pre-warmed at 37°C.
20.0 mg (actual weights between 20.0 mg and 20.9 mg) of “Acid Brown 432” were applied directly on the skin surface with a suitable spatula.
Every incubation time at room temperature under chemical hood [3 and 60 min.(± 5 min.) and 240 min.(± 10 min.)] had a separate plate containing two replicates of test item, two replicate of NC, two replicate for NSC and two replicate with killed cells for NSMTT.
Only the exposure time of 240 minutes had also two replicates of PC and two killed cells Negative Control.


Positive Control (PC) and Negative Control (NC)
A volume of 50 µL of PC (glacial acetic acid) and 50 µL of NC (NaCl 9 g/L) was applied on the skin surface by using a suitable pipette.


Rinsing
After the three exposure times, the EpiSkinTM SM units were removed and rinsed thoroughly with 25 mL of DPBS solution (2.0 mL/rinse) to remove the test item from the skin surface. The remaining DPBS was removed from the skin units by gently taping on absorbent paper and sweeping the surface with a cotton-bud.
The skin units were transferred to new plates prefilled with fresh pre-warmed (37°C) assay medium (2.0 mL/well) until also the last insert was rinsed.
At this time, the skin units were placed on absorbent paper to remove the assay medium. Then they were transferred into MTT.




MTT test
The units were transferred into wells filled with MTT solution (2.0 mL of 0.3 mg/mL MTT for each well), except for the units for the NSC control, which were filled with assay medium, and then all the units were incubated for three hours (± 15 min.) protected from light into the incubator at 37°C, 5% CO2, 95% humidify atmosphere.


Formazan extraction
At the end of MTT incubation (for each time of incubation), the formazan extraction was undertaken:
• Conical microtubes ”safe lock type” were labelled;
• The tissue units were placed on absorbent paper for 10-15 seconds to remove all the MTT and assay before placing them on the plate lid for biopsy.
• A disk of epidermis was cut from the unit using the biopsy punch and the epidermis was detached from its collagen matrix using forceps. Both parts (epidermis and collagen matrix) were turned on one another so that the topical side of the epidermis was in contact with the matrix, and placed into the labelled conical microtubes with 500 µL of acidified isopropanol. One tube corresponded to one well of the tissue culture plate.
• The tubes were capped and thoroughly mixed by using a vortex for 20 seconds to achieve a good contact of all the material with solvent.
• The tubes were then stored at room temperature overnight, protected from light.


Cell viability measurements (Day 2)

Following the formazan extraction, 2x200 µL samples from each tube were placed into the wells of a 96-well plate (labelled appropriately) and the OD (Absorbance/Optical Density) of the samples was read in a 96-well plate spectrophotometer at 570 nm using acidified isopropanol solution as the blank (6x200 µL).

Results and discussion

In vivo

Results

Irritation parameter:

other: mean relative tissue viability

Basis:

mean

Remarks:

negative control

Time point:

other: 4 hours

Score:

> 35

Irritant / corrosive response data:

The test item is not corrosive to skin being the mean relative viability after 4 hours of exposure ≥ 35% of the negative control.

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

During the preliminary test phases it was proved that ”Acid Brown 432”:
- was naturally colored and became coloured during the treatment, so it was MTT-reducer and interfered with MTT;
- affected the OD with a-specific MTT reduction, so the use of killed epidermis to evaluate the real OD was necessary.
- In contact with water, it showed ability to become coloured, so the quantification of NSC contribution was necessary.

After the main test, it was assessed that:

”Acid Brown 432”, after 3 minutes exposure had a mean tissue viability > 35% (103.59%), after 1 hour exposure had a mean tissue viability > 35% (94.78%) and only after 4 hours exposure, had mean tissues viability > 35%.(92.62%)
According to EU CLP, and,- on the basis of packing group/classification categories, the test item is Not corrosive.

All the assay Acceptance Criteria were satisfied, because:
• The mean OD value of the two negative control tissues for every incubation time was between 0.6 and 1.5 at 570 nm.
(0.946 after 3 minutes exposure; 1.023 after 60 minutes exposure and 0.860 after 240 minutes treatment, as reported in Tables 4, 8 and 12).
• The acceptable mean percentage viability range for positive controls was included
between 0 and 20%.
(In this case, the value was 4.19%, as reported in Table 14).
• In the range of 20-100% viability and for ODs ≥ 0.3, the difference of viability between the two tissue replicates did not exceed 30%.

For all the pre-incubation time and the MTT incubations, the temperature conditions were respected, according to the Internal SOP (37°C ± 1), with a mean of 37.60°C.
Also the humidity % was satisfied during that time with a mean of 95.29 %, while the CO2 signed by the display was always 5%, according to Internal SOP.
Since all the conditions for the validity of the test are satisfied, the test is valid and qualified.

Executive summary:

Tissue culture inserts of EpiSkin reconstructed human epidermis (two units/chemical) were treated with the test item, ”Acid Brown, and incubated for 3, 60 and 240 minutes at room temperature in order to identify the class of corrosivity. The test was performed according to OECD Guideline No. 431 “In vitroskin corrosion: reconstructed human epidermis test method” adopted on 26^thof September 2014.

Exposure of the test material was terminated by rinsing with Dulbecco’s phosphate buffered saline solution (DPBS).

The viability of each insert was assessed by incubating the tissues for 3 hours (±15 min.) with3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl bluetetrazolium bromide(MTT) solution in a specific incubator at, 5% CO₂, 95% relative humidity.

The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically at 570 nm (±25 nm).

 

NaCl (9 g/L saline) and Glacial Acetic Acid treated inserts were used as negative and positive controls, respectively.

 

For each treated tissue, viability was expressed as % compared to the negative control.

The test item is considered to be not corrosive to skin if the mean relative viability after 4 hours of exposure is ≥ 35% of the negative control.

The three incubation times (3, 60 and 240 minutes) are used to identify the class of corrosivity, according to the United Nations Globally Harmonized System (UN GHS) as adopted by the European Regulation (EC) No 127/2008 on classification labelling and packaging of substances and mixtures (CLP) Classification for the packing group.

 

 

In order to avoid to overvalue the optical density (OD) due to the Non Specific Color (NSC) and to the Non Specific3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl bluetetrazolium bromide(NSMTT), were performed two preliminary tests, one to check if test item interacted with MTT, and one to check the coloring potential.

Both the tests gave positive results, so that it was necessary the use of killed cells for binding the color.(the killed cells do not possess metabolic activity, but they absorb and bind the test item like viable tissues), and the use of vital cells for the quantification of NSC(Non Specific Color).

In fact, the true viability %, is the result of the subtraction of NSC and NSMTT values to the viability % obtained in each application times. In this case, these additional tests were necessary, because it was verified that the test item had interactions.

According to EU CLP, and,- on the basis of packing group/classification categories, the Acid Brown 432 is Not corrosive.