2,3,5,6-tetrachloropyridine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 June 2009 to 29 June 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The skin irritation potential of the test material is evaluated using the EPISKINTM reconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours (1, 2, 3, 4). The principle of the assay is based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 hour post exposure incubation period is also determined for test materials which are found to be borderline non irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.
The EPISKINTM model is a three-dimensional reconstituted human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13 day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,3,5,6-tetrachloropyridine

EC Number:

219-283-9

EC Name:

2,3,5,6-tetrachloropyridine

Cas Number:

2402-79-1

Molecular formula:

C5HCl4N

IUPAC Name:

2,3,5,6-tetrachloropyridine

Details on test material:

Batch number XF09160201 of purity = 99.4%

Test animals

Details on test animals or test system and environmental conditions:

Not applicable

Test system

Amount / concentration applied:

Not applicable

Duration of treatment / exposure:

Not applicable

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

PROCEDURE

Pre-Test

Assessment of Direct Test Material Reduction of MTT

MTT dye metabolism, cell viability assay

The MTT assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a purple formazan dye by mitochondrial succinate dehydrogenase in viable cells.

Assessment of Direct Test Material Reduction of MTT

One limitation of the assay is possible interference of the test substance with MTT. A test substance may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test substance is only a problem, if at the time of the MTT test (after rinsing) there is still sufficient amounts of the test substance present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by the procedure described below.

Test for Direct MTT Reduction

As specified, a test substance may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, each test substance is checked for the ability to directly reduce MTT according to the following procedure:
10 mg of the test material was added to 2 ml of a 0.3 mg/ml MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37ºC, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test substance turns blue/purple, the test substance is presumed to have reduced the MTT.

Pre-Incubation (Day 0: tissue arrival)

2 ml of maintenance medium, warmed to approximately 37ºC, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test material and each control material. The tissues were incubated at 37ºC, 5% CO2 in air for at least 24 hours.

Main Test

Application of Test Material and Rinsing (Day 1)

2 ml of maintenance medium, warmed to approximately 37ºC, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test material for an exposure period of 15 minutes. The test material was applied topically to the corresponding tissues ensuring uniform covering. 10 ± 2 mg of the test material was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl sterile distilled water to improve contact between the solid test material and the epidermis. Triplicate tissues treated with 10 µl of PBS served as the negative controls and triplicate tissues treated with 10 µl of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control material the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7 minutes contact time the SDS solution was re spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 ± 0.5 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing PBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material. The rinsed tissues were transferred to the second column of 3 wells containing 2 ml of maintenance medium in each well. The rinsed tissues were incubated at 37ºC, 5% CO2 in air for approximately 42 hours.

MTT Loading/Formazan Extraction (Day 3)

Following the 42-hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 ± 2 minutes to homogenise the released mediators in the maintenance medium. 1.6 ml of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and store in a freezer at 14 to 30ºC for possible inflammatory mediator determination.
2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37°C, 5% CO2 in air. At the end of the 3 hour incubation period each tissue was placed onto absorbent paper to dry. The tissues were examined and the degree of MTT staining evaluated (qualitative evaluation of cell viability) using the MTT Visual Scoring scheme. Following qualitative evaluation of tissue viability a total biopsy of the epidermis was made using the EPISKINTM biopsy punch.The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 ml micro tubes containing 500 µl of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6)

At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.
For each tissue, duplicate 200 µl samples were transferred to the appropriate wells of a pre labelled 96 well plate. 200 µl of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: MTT reduction

Value:

115.3

Remarks on result:

other:

Remarks:

Time point: 15 minutes. Remarks: Score given is the relative mean viability of the test material treated tissues in % terms . (migrated information)

In vivo

Irritant / corrosive response data:

RESULTS

Direct MTT Reduction

The MTT solution containing the test material did not turn blue/purple which indicated that the test material did not directly reduce MTT.

Test Material, Positive Control Material and Negative Control Material

The individual and mean OD540 values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.
The relative mean viability of the test material treated tissues was 115.3% after a 15 minute exposure.
The qualitative evaluation of tissue viability is given in Table 2.
Following the 15-minute exposure the test material treated tissues appeared blue which was considered indicative of viable tissue.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was ≤40% relative to the negative control treated tissues and the Standard Deviation (SD) value of the % viability was ≤20%. The positive control acceptance criterion was therefore satisfied.
The mean OD540 for the negative control treated tissues was ≥0.6 and the SD value of the % viability was ≤20%. The negative control acceptance criterion was therefore satisfied.

CONCLUSION

The test material was considered to be Non-Irritant.

Any other information on results incl. tables

Table1              Mean OD₅₄₀Values and % Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material	OD540of tissues	Mean OD540of triplicate tissues	± SD of OD540	Relative individual tissue viability %	Relative mean % viability	± SD of % viability
Negative Control Material	0.914	0.896	0.026	102.0	100*	2.8
	0.908			101.3
	0.867			96.8
Positive Control Material	0.143	0.126	0.040	16.0	14.1	4.5
	0.155			17.3
	0.081			9.0
Test Material	1.052	1.033	0.063	117.4	115.3	7.0
	1.085			121.1
	0.963			107.5

 

* = The mean viability of the negative control tissues is set at 100%

Table2              Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material	Tissue 1	Tissue 2	Tissue 3
Negative Control Material	-	-	-
Positive Control Material	+	+	+
Test Material	-	-	-

MTT visual scoring scheme
-          =         blue tissue (viable)
+         =         blue/white tissue (semi-viable)
++       =         tissue is completely white (dead)

 

*= The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: According to the principles of the test method

Conclusions:

The substance is considered to be non-irritant.

Executive summary:

The skin irritation potential of the test material was evalauted using the EPISKIN^TMreconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The substance was concluded to be non-irritant according to the conditions of the test.