Ethanol, 2-[(3-nitropyrazolo[1,5-a]pyridin-2-yl)oxy]-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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

Test material

Test material information

Constituent 1

Reference substance name:

2-[(3-nitropyrazolo[1,5-a]pyridine-2-yl)oxyethanol

IUPAC Name:

2-[(3-nitropyrazolo[1,5-a]pyridine-2-yl)oxyethanol

Constituent 2

Reference substance name:

IMEXINE stage 4 R0056895A

IUPAC Name:

IMEXINE stage 4 R0056895A

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Details on test animals or test system and environmental conditions:

human reconstructed epidermis (tissues).The EpiskinTM model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstructed from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra structure and is functionally equivalent to human in vivo epidermis.

Test system

Amount / concentration applied:

Test item quantity applied to each tissue: 20 mg ± 2 mg.
The test item was spread over the whole tissue surface without damaging the tissue sample.
As the test item was a solid, 100 µL of 0.9% NaCl were applied over the test item to ensure good contact with the epidermis.
For the negative and positive controls, since they can be sampled using a pipette, a volume of 50 µL was applied evenly to the surface of each tissue, taking care to spread them over the whole tissue surface area without damaging the tissue sample.

Negative control
Name: 0.9% NaCl.
The origin and batch number of the negative control used are recorded in the study files.

Positive control
Name: glacial acetic acid.
The origin and batch number of the positive control used are recorded in the study files.

Details on study design:

1.Preliminary test
1.1Test for direct MTT reduction with the test item
As a test item may directly reduce MTT, thus mimicking mitochondrial succinate dehydrogenase activity, it is necessary to test the ability of the test item to directly reduce MTT before performing the main test. This property of the test item would only be a problem if, at the time of the MTT viability test (after rinsing), remained a sufficient amount of test item present on or in the tissue. In this case, the true metabolic MTT reduction and the false direct MTT reduction should be differentiated and quantified.
To identify any test item interference, the following preliminary test was performed: 20 mg of test item was added to 2 mL of a 0.3 mg/mL freshly prepared MTT solution. The mixture was incubated in darkness at +37°C for 3 hours (± 5 minutes) under continuous stirring. A negative control was tested concurrently by adding 50 µL of water for injection to 2 mL of a 0.3 mg/mL freshly prepared MTT solution.

If the MTT solution color turned blue/purple when compared to the negative control (complete or partial discoloration), the dose formulation was presumed to reduce MTT. In this case additional controls were performed on water killed tissues in parallel to the main test (performed on viable tissues).
1.2Test for the detection of the coloring potential of the test item
The intrinsic color or the ability of the test item to become colored in contact with water (simulating a tissue humid environment) was evaluated by adding 10 mg of test item to 90 µL of water for injection in a transparent recipient. After 1 hour of mixing, the coloration was checked.
If a colored solution was detected, additional controls were performed on viable tissues in parallel to the main test for the evaluation of the non specific OD. Otherwise, the test item was considered as a common chemical.

2 Main test
One 12-well plate was used for each of the three exposure times (3 minutes, 1 hour and 4 hours) for test item-treated tissues.
Positive and negative controls were placed on separate plates.
Test item, and negative and positive controls were applied on duplicate tissues.

2.1 Pre-incubation of the tissues on their day of arrival
A volume of 2 mL maintenance medium, pre-warmed (at 37°C), was added to 2 wells per 12 well plate as follows: duplicate wells for each test item and negative control exposure time and for the 4 hour positive control exposure time.
EpiskinTM kits were opened and one tissue was transferred into each maintenance medium pre-filled well. All 12-well plates were then placed into a cell incubator (37°C, 5% CO2, >95% humidity) for a 1 hour to 48 hours pre-incubation step before treatment.

2.2 Treatment of tissues
A volume of 2 mL assay medium, pre-warmed (at 37°C), was added to 2 wells per 12 well plate as follows: duplicate wells for each test item and negative control exposure time and for the 4 hour positive control exposure time.
The EpiskinTM tissues were removed from the incubator and one tissue was transferred into each assay medium pre-filled well.

The test item, positive control or negative control dose formulations were applied on each designated tissue.
The lids were replaced on each plate before incubation at room temperature as follows: positive control for 4 hours (± 10 minutes); test item and negative control for 3 minutes (± 5 seconds), 1 hour (± 5 minutes) and 4 hours (± 10 minutes).

For MTT reducing test items, duplicate test item-treated water-killed tissues were prepared in a 12-well plate and incubated under the same conditions as test item-treated viable tissues. Duplicate 0.9% NaCl treated water-killed tissues were prepared in a 12-well plate and incubated for 4 hours.

Tissues were processed (application and rinsing) in the same order and at regular time-intervals so the tissue exposure times were equal.

2.3 Rinsing of tissues
For all treated tissues [test item-treated, positive and negative controls, and treated water killed tissues (if applicable)], at the end of the designated incubation period each tissue insert was removed from the well of the treatment plate and rinsed with D-PBS.
Rinsing was achieved by gently filling and emptying each tissue insert 12 times with 2 mL D-PBS and the surface of each tissue was swept with a cotton-bud to gently remove any residual dose formulation. The inserts were then placed in assay medium pre-filled well (2 mL).

2.4 MTT viability assay
Two empty wells of each 12-well plate were filled with 2 mL MTT solution (0.3 mg/mL), and the corresponding tissues were placed in these wells. Each plate was protected from light and incubated for 3 hours (± 15 minutes) at +37°C, 5% CO2 in a humidified incubator.

At the end of the MTT incubation period, the underside of each tissue culture insert was blotted. Then the tissues were removed from its plastic insert using a biopsy punch. Any tissue discoloration was evaluated with the naked eye.

Thereafter, for each tissue, the epidermis was separated from the collagen matrix. Both parts (epidermis and collagen matrix) were put into a stoppered plastic tube and 0.5 mL acidified isopropanol were then added to each tube. After vortexing, each tube was protected from light and left at room temperature overnight to extract the formazan (reduced MTT) from the MTT-loaded tissues.

2.5 Optical density measurements
At the end of the extraction period, each tube was vortexed and briefly centrifuged avoiding the interference of cell fragments with the absorbance readings.

Each tube was used to fill 2 consecutive wells of a 96-well plate with 200 µL of extract per well. One 96 well plate was used for the negative and positive controls (placed at opposite ends of the plate), and a separate 96-well plate was used for the test item dose formulation (the extracts obtained after all exposure times were placed on the same plate) and, if applicable, the extracts from the treated and 0.9% NaCl treated water-killed tissues were each placed on an additional 96-well plates.

For each 96-well plate, the average Optical Density value (OD) of 4 wells containing 200 µL of acidified isopropanol only was used as the blank.

The OD was measured at 570 nm using a plate reader.
The test complies when:
 the mean cOD of the negative control is between 0.600 and 1.500,
 relative mean viability of the positive control is ≤ 20% of the relative mean viability of the negative control,
 in the range 20-100% viability and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30%.

Results and discussion

In vivo

Irritant / corrosive response data:

non corrosive

Any other information on results incl. tables

1      PRELIMINARY TESTS

1.1      Test for direct MTT reduction with the test item

During the preliminary test, the MTT solution color turned blue/purple when compared to the negative control, the dose formulation is presumed to reduce MTT. As a result, additional controls were performed on water-killed tissues in parallel to the main test (performed on viable tissues). The MTT‑reducing test item and negative control were applied to two water-killed tissues for each exposure time.

 

1.2      Test for the detection of the coloring potential of the test item

During the preliminary test, the solution did not become colored, the test item is presumed not to stain the tissue. As a result, no additional controls were performed on viable tissues in parallel to the main test.

 

 

2      MAIN TEST

 

2.1   Evaluation of the coloration of tissues at the end of the MTT incubation period

The qualitative evaluation of the MTT staining was performed with the naked eye and all treated tissues appeared blue which was considered indicative for viable tissues.

 

1.2.3      Evaluation of the MTT results

All of the acceptance criteria for the negative and positive controls were fulfilled, therefore the study was considered to be valid.

 

The mean blank OD values (mean OD_blank) are calculated from the four replicates on each plate.

Then, for each tissue, the mean OD_blankis subtracted from individual tissues OD and the background corrected mean OD values (mean cOD) are calculated.

For each exposure duration, the corrected mean OD of the two negative controls (mean cOD_NC) corresponds to 100% viability and is used as a reference.

For the tissues treated with the test item or positive control, the relative mean viabilities are expressed as percentages of the reference viability and are calculated as follows:

             Relative mean viability = (mean cOD_{TI or PC}/ mean cOD_NC) x 100.

 

with        mean cOD_TI: corrected mean OD of the three tissues treated with test item,

             mean cOD_PC: corrected mean OD of the three positive controls.

 

The relative mean viabilities of the test item-treated tissues were:

.            3 minutes exposure: 105%,

.            1 hour exposure: 100%,

.            4 hours exposure: 103%.

 

As the mean viability was≥35% at each exposure time,the results met the criteria for a non corrosive response.

Applicant's summary and conclusion

Interpretation of results:

other: non corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the experimental conditions of this study, the test item, R0056895A, tested in its original form, is considered to be non corrosive to the reconstructed skin model.

According to these results, the classification of the test item should be the followingconsidered as: non corrosive (Directive 67/548/EEC, UN 2009 and Regulation 1272/2008/EEC.

Executive summary:

The objective of this study was to evaluate the corrosive potential of the test item, R0056895A, using the

EpiskinTM reconstructed skin model.

This study was conducted in compliance with CiToxLAB France’s standard operating procedures and the

principles of Good Laboratory Practices.

Preliminary tests were performed to detect the ability of the test item to interfere with cell viability

measurements by directly reducing MTT.

Following the preliminary test, the corrosive potential of the test item was tested in the main assay.

Test item, and negative and positive controls were applied on duplicate tissues and incubated at room

temperature as follows: positive control for 4 hours (± 10 minutes); test item and negative control for 3 minutes (± 5 seconds), 1 hour (± 5 minutes) and 4 hours (± 10 minutes).

At the end of the designated incubation period, each tissue insert was rinsed with D-PBS and put into 2 mL assay medium pre-filled well. Then, two empty wells of each 12-well plate were filled with 2 mL MTT solution (0.3 mg/mL), and the corresponding tissues were placed in these wells. Each plate was protected from light and incubated for 3 hours (± 15 minutes) at +37°C, 5% CO2 in humidified atmosphere.

At the end of the MTT incubation period, the tissues were removed from its plastic insert. Any tissue discoloration was evaluated with the naked eye (discolored surface area and intensity).

Then, for each tissue, the epidermis was separated from the collagen matrix and both parts were put into acidified isopropanol overnight to extract the formazan (reduced MTT) out of the MTT-loaded tissues.

At the end of the extraction period, the optical density of each extract was measured at 570 nm.

Relative viability values were calculated for each tissue and expressed as percentages of the negative control tissues viability which was set at 100% (reference viability).

In the preliminary tests, the test item was found not to have direct MTT reducing properties or coloring potential.

In the main test, all acceptance criteria for the negative and positive controls were fulfilled. The study was therefore

considered to be valid.

At the end of the MTT incubation period, any tissue discoloration was evaluated with the naked eye.

The blue discoloration of the test item-treated tissues following the 3 minutes, 1 hour and 4 hours exposure periods was representative of viable tissue.

The relative mean viabilities of the test item-treated tissues were:

. 3 minutes exposure: 98%,

. 1 hour exposure: 98%,

. 4 hours exposure: 100%.

As all the mean viabilities were ≥ 35%, the results met the criteria for a non corrosive response.