Registration Dossier

Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
other: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 April 2010 and 30 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to generally valid and/or internationally accepted testing guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline for the Testing of Chemicals No. 431 "In Vitro Corrosion: Human Skin Model Test" (adopted 13 April 2004)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Propylene glycol ether (heavy stream)
- Physical state: dark brown liquid
- Lot/batch No.: 000000857730
- Date received: 29 January 2010
- Expiration date of the lot/batch: not supplied
- Storage condition of test material: room temperature in the dark
- Other: The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor.

Test animals

Species:
other: reconstituted human epidermis
Details on test animals and environmental conditions:
Not applicable for in vitro test.

Test system

Type of coverage:
other: not applicable for in vitro test
Preparation of test site:
other: not applicable for in vitro test
Vehicle:
other: not applicable for in vitro test
Controls:
other: not applicable
Amount / concentration applied:
See free text in "Details on study design" section below.
Duration of treatment / exposure:
15 minutes direct exposure. See free text in "Details on study design" section below.
Observation period:
See free text in "Details on study design" section below.
Number of animals:
Not applicable to an in vitro study.
Details on study design:
MTT dye metabolism, cell viability assay:

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

Assessment of Direct Test Material Reduction of MTT:

A test substance may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test substance is a problem only if at the time of the MTT test (after rinsing) there are 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 as follows:

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:

50 microliters of the test material was added to 2.2 ml of a 0.3 mg/ml MTT solution freshly prepared in assay medium. The solution was incubated in the dark at room temperature 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):

2.2 ml of maintenance medium, warmed to approx. 37 deg C, was pipetted into two wells of the first column of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12-well plate was used for the test material and each control material. The tissues were incubated at 37 deg C, 5% CO2 in air for approximately 24 hours after which time the medium was replaced with 2.2 ml of fresh maintenance medium and the tissues further incubated at 37 deg C, 5% CO2 in air overnight.

Main Test:

Application of the Test Material and Rinsing (Day 2):

2.2 ml of maintenance medium, warmed to approx. 37 deg C, was pipetted into 2 wells of the second and third columns of the 12-well plate. The tissues were transferred into the second column.

Duplicate tissues were treated with the test material for periods of 3, 60 and 240 minutes. Duplicate tissues were treated with the positive and negative control materials for an exposure period of 240 minutes. 50 microliters of the test material was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. Duplicate tissues, treated with 50 microliters of 0.9% w/v sodium chloride solution served as negative controls. Duplicate tissues, treated with 50 microliters of glacial acetic acid served as positive controls. The treated tissues were kept in the biological safety cabinet at room temperature for the appropriate exposure period.

At the end of each 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 approx. 40 seconds using a constant soft stream of PBS to gently remove any residual test material. Each rinsed tissue was placed into a third column of the 12-well plate until tissues were rinsed.

2.2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium, was pipetted into two wells of the forth column of each 12 well plate. The tissues were transferred to the MTT filled wells. The tissues were incubated for 3 hours +/- 5 minutes at room temperature in a biological safety cabinet ensuring the plates were protected from light. 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). Following qualitative evaluation of tissue viability a total biopsy of the epidermis was made using the EPISKIN(TM) biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 ml micro tubes containing 850 microliters of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 3):

At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution. For each tissue, duplicate 200 microliter samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 microliters 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.

Interpretation of Results:

Quantitative MTT Assessment (percentage tissue viability):

The corrosivity of the test material was predicted from the relative mean tissue viabilities obtained after the 3, 60 and 240-minute treatments, compared to the mean of the negative control treated tissues (n=2). The relative mean viabilities were calculated in the following way: Relative mean = (mean OD540 of test material/mean OD540 of negative control) x 100 viability (%).

Classification of corrosivity potential was based upon relative tissue viabilities for each exposure time according to the prediction model (Table 1):

Quality criteria:

The results of the assay are considered acceptable if the following assay acceptance criterion is achieved:

Positive Control:

The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was 0 to 20 % relative to the negative control treated tissues following the 240-minute exposure period.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: Tissue viability (% of negative control)
Basis:
mean
Time point:
other: 240 minutes
Score:
12.4
Irritation parameter:
other: Tissue viability (% of negative control)
Basis:
mean
Time point:
other: 60 minutes
Score:
78.3
Irritation parameter:
other: Tissue viability (% of negative control)
Basis:
mean
Time point:
other: 3 minutes
Score:
78.3
Irritant / corrosive response data:
Direct MTT Reduction:

The MTT solution containing the test material turned blue/purple, which indicated that the test material directly reduce MTT. The assay was therefore performed in parallel on viable and water-killed tissues.

Mean OD540 values and viabilities for the negative control, positive control material and test material were calculated.

Quality Criteria:
The relative mean tissue viability for the positive control treated tissues was 8.5% relative to the negative control treated tissues following the 240-minute exposure period. The positive control acceptance criterion was therefore satisfied.

The qualitative evaluation of tissue viability (MTT uptake visual evaluation) is shown in Table 2.

Any other information on results incl. tables

Table 2: Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material  Exposure Period  Tissue 1  Tissue 2 
Negative Control Material  240 Minutes 
Positive Control Material  240 Minutes  ++  ++ 
Test Material  240 Minutes  -
   60 Minutes  -  -
   3 Minutes  -  -

MTT visual scoring scheme

- = blue tissue (viable)

+ = blue/white tissue (semi-viable)

++ = tissue is completely white (dead)

Applicant's summary and conclusion

Interpretation of results:
corrosive
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material was classified as corrosive to the skin according to EU labelling regulations Commission Directive 2001/59/EC. The symbol "C", the indication of danger "Corrosive" and the risk phrase R34 "Causes Burns" are therefore required. The UN packing group III is also required.