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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 January 2011 to 24 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was conducted using an alternative testing method (EPISKIN reconstructed human epidermis model) considered to provide valid data for the skin irritation/corrosion endpoint. The study was also performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011

Materials and methods

Principles of method if other than guideline:
The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item 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 item treated tissue relative to the negative controls. The concentration of the inflammatory mediator IL-1alpha in the culture medium retained following the 42-hour post-exposure incubation period is also determined for test items which are found to be borderline non-irritant based on the MTT reduction endpoint. This complementary endpoint will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

The EPISKIN model is a three dimensional reconstructed 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 a 13-day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification: 3-trifluoromethylaniline
Description: Extremely pale yellow liquid
Batch Number: 101009
Purity: 99.66 %
Date Received: 25 October 2010
Storage Conditions: Room temperature in the dark

Test system

Details on study design:
PREPARATION OF TEST ITEM
The test item was used as supplied.

NEGATIVE AND POSITIVE CONTROL ITEMS
Dulbecco's Phosphate Buffered Saline (PBS) with Ca++ and Mg++ was used as the negative control.
Sodium Dodecyl Sulphate (SDS) 5% w/v was used as the positive control.

PREPARATION OF NEGATIVE AND POSITIVE CONTROL ITEMS, MTT AND ACIDIFIED ISOPROPANOL
The negative control item was used as supplied.
The positive control item was prepared as a w/v aqueous dilution.
A 3 mg/mL MTT stock solution was prepared in PBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
A 0.04 N concentration of hydrochloric acid in Isopropanol was prepared when required.

EPISKIN MODEL KIT
Date Received: 18 January 2011.

PROCEDURE
PRE-TEST
ASSESSMENT OF DIRECT TEST ITEM 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.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic reduction and the false direct MTT reduction can be differentiated and quantified.

TEST FOR MTT REDUCTION
Each test item is checked for the ability to directly reduce MTT according to the following procedure:
10 uL of the test item 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 37 degrees celsius, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT.

PRE-INCUBATION (DAY 0: TISSUE ARRIVAL)
2 mL of maintenance medium, warmed to approximately 37 degrees celsius, 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 ite, amd each control item. The tissues were incubated at 37 degrees celsius, 5% CO2 in air overnight.

MAIN TEST
APPLICATION OF TEST ITEM AND RINSING (DAY 1)
2 mL of maintenance medium, warmed to approximately 37 degrees celsius, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 uL of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 uL of PBS served as the negative controls and triplicate tissues treated with 10 uL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item 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 approximately 15 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 item. 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 37 degrees celsius, 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 approximately 15 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 stored in a freezer at -14 to -30 degrees celsius 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 degrees celsius, 5% CO2 in air. At the end of the 3-hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using EPISKIN 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 uL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughlly in a vortex mixer. The tubes were refrigerated at 1 to 10 degrees celsius until Day 6 of the experiment, allowing 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 thoroughlly on a vortex mixer to produce a homogenous coloured solution.
For each tissue, duplicate 200 uL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 uL 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:
Value:
5
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15-min exposure; 42-hour post-exposure incubation period. Reversibility: no data. (migrated information)

Any other information on results incl. tables

DIRECT MTT REDUCTION

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

TEST ITEM, POSITIVE CONTROL ITEM AND NEGATIVE CONTROL ITEM

The individual and mean OD540 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are presented in Table 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also presented in Table 1.

Table 1. Mean OD540 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item.

 Item  OD540 of Tissues  Mean OD540 of Triplicate Tissues  SD of OD540  Relative Individual Tissue Viability (%)  Relative Mean Vaibility (%) SD of Relative Mean Viability (%) 
 Negative Control Item  0.735 0.720 0.017  102.1, 100.3        100 2.3       
 0.722
 0.702  97.5
 Positive Control Item  0.055 0.046 0.011         7.6        6.4 1.5       
 0.050  6.9
 0.034  4.7
Test Item  0.072 0.036 0.031  10.0        5.0        4.3
 0.016  2.2
 0.021  2.9

The relative mean viabilityh of the test item treated tissues was 5.0% after a 15 -minute exposure period.

QUALITY CRITERIA

The relative mean tissue viability for the positive control treated tissues was less than or equal to 40% relative to the negative control treated tissues and the SD value of the percentage viability was less than or equal to 18 %. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was greater than or equal to 0.6 and the SD value of the percentage viability was less than or equal to 18 %. The negative control acceptance criterion was therefore satisfied.

The SD calculated form individual percentage tissue viabilities of the three identically treated tissues was less than or equal to 18 %. The test item acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be an irritant (I).
The test item was also classified as irritant according to Commission Directive 2001/59/EC. The symbol "I", the indication of danger "irritant" and the risk phrase R38 "irritating to skin" are therefore required.