Reaction mass of 3-methyl-1-octylpyridinium chloride and 4-methyl-1-octylpyridinium chloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 3-methyl-1-octylpyridinium chloride and 4-methyl-1-octylpyridinium chloride

EC Number:

941-484-1

Molecular formula:

not possible

IUPAC Name:

Reaction mass of 3-methyl-1-octylpyridinium chloride and 4-methyl-1-octylpyridinium chloride

Test material form:

liquid: viscous

Test animals

Species:

human

Strain:

other: EPISKIN-SM™ tissue (SkinEthic)

Details on test animals or test system and environmental conditions:

The EPISKIN-SMTM tissues are provided as kits (SkinEthic), consisting of the following components
relevant for this study:
1x EPISKIN-SM™ plate containing 12 reconstructed epidermis units (area: 0.38 cm2); each
reconstructed epidermis is attached to the base of a tissue culture insert with an O-ring set and
maintained on nutritive agar for transport (Lot: 14-EKIN-034)
1x 12-well assay plate
1x flask of sterile maintenance medium (basic medium for incubations, Lot: 14-MAIN3-039)
1x flask of sterile assay medium (basic medium for use in MTT assays, Lot: 14-ESSC-037)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: positive and negative control tissues

Amount / concentration applied:

Dose Groups
1. Negative control 50 μL 0.9% NaCl solution
2. Positive control 50 μL glacial acetic acid
3. Test Item 50 ± 3 μL (undiluted)

MTT stock solution will be diluted 1 + 9 with DMEM-based medium (final concentration 0.3 mg/mL)

Duration of treatment / exposure:

3 minutes, 60 minutes and 4 hours

Number of animals:

The test was performed on a total of 6 tissues for each test item and for the negative control, 2 replicates for each treatment period (3 min, 60 min and 4 h exposure time). For the positive control the test was performed with 2 replicates for the 4 h exposure time.

Details on study design:

4 h experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. Then the 12-well plate(s) were incubated for 4 h ± 10 min. at room temperature.

60 min. experiment: the tissues were treated with the test item and the negative control in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. Then the 12-well plate(s) were incubated for 60 min. ± 5 min. at room temperature.

3 min. experiment: the tissues were treated with the test item and the negative control in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of e.g. 30 sec. was kept between dosing. After 3 min. of application, with forceps, the first insert was removed from the 12-well plate. Using e.g. a wash bottle the tissue was gently rinsed about 15 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The surface was swept with a cotton bud. The insert was placed in a prepared 2-well “holding plate“ containing pre-warmed assay medium per well. All inserts were treated in the same manner.
Then the inserts were transferred into a prepared 12-well “MTT assay plate“ containing prewarmed MTT solution. The plate was incubated for 3 h ± 15 min. at 37 °C, 5.0% CO2 / 95% air.

60 min and 4 h experiment: after 60 min. / 4 h application, with forceps, the first insert was removed from the 12-well plate. Using e.g. a wash bottle the tissue was gently rinsed about 15 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The surface was swept with a cotton bud. The insert was placed in a prepared 12-well “holding plate“ containing pre-warmed assay medium per well. All inserts were treated in the same manner.
Then the inserts were transferred into a prepared 12-well “MTT assay plate“ containing prewarmed MTT solution. The plate was incubated for 3 h ± 15 min. at 37 ± 1 °C, 5.0% CO2 / 95% air.

3 min., 60 min. and 4 h experiment: After the 3 h MTT incubation period the tissues were placed on blotting paper to remove excess liquid. Afterwards a total biopsy of the epidermis by using the biopsy punch was performed and the epidermis was separated from the collagen matrix with the aid
of forceps. Both parts (epidermis and collagen matrix) were transferred into suitable tubes and acidic isopropanol were added. Then the tubes were mixed on a vortex mixer. Extraction was carried out at room temperature overnight, protected from light.
At the end of the formazan extraction period the tubes were mixed by vortexing until solution color became homogeneous.
If any visible cell/tissue fragments were in suspension, the tubes were centrifuged at 500 rpm to eliminate the fragments and avoid possible interference with the absorbance readings.
Per each tissue aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm without reference wavelength in a plate spectrophotometer.

Results and discussion

Any other information on results incl. tables

3 min.

Name	Negative Control		Test Item
Tissue	1	2	1	2
absolute OD570- values (raw data)	0.966	0.976	0.885	0.910
	0.944	0.960	0.882	0.888
mean OD570(mean of 2 aliquots)	0.955	0.968	0.884	0.899
mean OD570(blank corrected)	0.912	0.926	0.841	0.857
total mean OD570(mean of 2 replicate tissues, blank corrected)	0.919*		0.849
relative tissue viability [%]	99.3	100.7	91.5	93.2
mean relative tissue viability [%]	100		92
difference of relative tissue viability [%]***	1.5		1.6

*corrected mean OD570of the negative control corresponds to 100% absolute tissue viability.

***difference between each two replicates is≤30% (in the range of 20 – 100% viability and for

ODs > 0.3).

60 min.

Name	Negative Control		Test Item
Tissue	1	2	1	2
absolute OD570- values (raw data)	0.901	0.960	0.645	0.635
	0.890	0.955	0.645	0.637
mean OD570(mean of 2 aliquots)	0.895	0.957	0.645	0.636
mean OD570(blank corrected)	0.853	0.915	0.603	0.594
total mean OD570(mean of 2 replicate tissues, blank corrected)	0.884*		0.598
relative tissue viability [%]	96.5	103.5	68.2	67.2
mean relative tissue viability [%]	100		68
difference of relative tissue viability [%]***	7.0		1.0

*corrected mean OD570of the negative control corresponds to 100% absolute tissue viability.

***difference between each two replicates is≤30% (in the range of 20 – 100% viability and for

ODs > 0.3). 4 h

Name	Negative Control		Test Item		Positive Control
Tissue	1	2	1	2	1	2
absolute OD570 -values (raw data)	0.922	0.842	0.238	0.238	0.077	0.061
	0.894	0.829	0.227	0.226	0.076	0.062
mean OD570(mean of 2 aliquots of 1 tissue)	0.908	0.835	0.232	0.232	0.076	0.061
mean OD570(blank corrected)	0.866	0.793	0.190	0.189	0.034	0.019
total mean OD570(mean of 2 replicate tissues, blank corrected)	0.829*		0.190		0.026
relative tissue viability [%]	104.4	95.6	22.9	22.8	4.1	2.3
mean relative tissue viability [%]	100		23		3**
difference of relative tissue viability [%]***	8.8		0.1		1.8

*corrected mean OD570of the negative control corresponds to 100% absolute tissue viability.

**mean relative tissue viability of the two positive control tissues of the 4 h treatment period is ≤ 20%.

***difference between each two replicates is ≤ 30% (in the range of 20 – 100% viability and for ODs > 0.3).

Applicant's summary and conclusion

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Remarks:

Migrated information

Conclusions:

In this study under the given conditions the test item showed corrosive effects. The relative mean tissue viability after 4 h treatment was decreased below 35%. Additionally, the relative mean tissue viability was decreased to less than 35% after 60 min. treatment. The test item is therefore
classified as “corrosive“ in accordance with optional UN GHS sub-category 1B/C.