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Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guideline 431.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Qualifier:
according to
Guideline:
other: OECD guideline 431 (2004)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Reaction mass of α-Butyl-ω-hydroxy-poly(oxy(methyl-1,2-ethanediyl)) (Polypropylene glycol monobutyl ether) and Tetramethyl-3,6,9,12-tetraoxahexadecan-1-ol (Tetrapropylene glycol n-butyl ether).

Test animals

Species:
human
Strain:
other: EpiDerm Skin Model (EPI-200)
Details on test animals and environmental conditions:
not applicable - in vitro assay

Test system

Type of coverage:
other: in vitro assay
Preparation of test site:
other: in vitro assay
Vehicle:
unchanged (no vehicle)
Controls:
other: in vitro assay
Amount / concentration applied:
Based upon MatTek Corporation’s recommended procedure, 50 μl of undiluted test material (liquids) were prepared and added on top of the skin tissues.
Details on study design:
The EpiDerm Skin Model (EPI-200). The model consisted of normal, human-derived epidermal kerotinocytes that were cultured to form an in vitro multilayered, highly differentiated model of the human epidermis. It consisted of organized basal, spinous, and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues were cultured on polycarbonate membranes of 10 mm cell culture inserts. Based upon MatTek Corporation’s recommended procedure, 50 μl of undiluted test material (liquids) were prepared and added on top of the skin tissues. The pH of the liquid test material was determined using both pH paper or pH meter. The test material(s) were administered by topical application to the EpiDerm skin tissue.

The skin tissues were placed in a refrigerator (4ºC) the day they were received. The next day, at least one hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 ml DMEM medium per well. The medium was replaced with fresh DMEM medium just before test compound was applied. The test was performed on a total of four tissues per test substance in addition to a negative and positive control compound. Two tissues were used for a three minute exposure to the test compound, and two tissues were used for a one hour exposure. Fifty μl of the undiluted test substance were added into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μl Milli-Q water (negative control) or 50 μl 8N KOH (positive control). Following the respective exposure period, the tissues were carefully washed with phosphate buffered saline (PBS) to remove residual test substance. Rinsed tissues were kept in 24 well plates on 300 μl DMEM medium and placed into a humidified (37ºC, 5% CO2) incubator until all tissues (12 application time, including controls) were dosed for the particular period and rinsed. The DMEM medium was replaced by 300 μl MTTmedium and tissues were incubated for 3 hr at 37°C, 5% CO2. After incubation the tissues were washed with PBS and indicator formazan was extracted with 2 ml isopropanol over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm (OD570) in triplicate with a VMax® Microplate Reader (Molecular Devices).

According to the manufacture‘s instruction, a killed control test was performed to assess whether residual test substance (TPnB-H) was acting to directly reduce the MTT. To test for residual test article MTT reduction, a killed tissue (Freeze/Thaw method) was treated with the test article (TPnB-H) in the same manner as for the viable tissue. A single killed control treated with water (negative control killed control) was tested in parallel since a small amount of MTT reduction was expected from the residual NADH and associated enzymes within the killed tissue. If little or no MTT reduction was observed in the test article-treated killed control, the MTT reduction observed in the test article-treated viable tissue may be ascribed to the viable cells. If there was appreciable MTT reduction in the treated killed control (relative to the amount in the treated viable tissue), the mean raw absorbance will be corrected for the amount of MTT reduced directly by the test material residue to obtain a final corrected OD570 value.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: mean viability
Basis:
other: in vitro assay
Time point:
other: 3 minutes
Score:
> 50
Remarks on result:
other: more than 50% mean viability
Irritation parameter:
other: mean viability
Basis:
other: in vitro assay
Time point:
other: 1 hour
Score:
> 15
Remarks on result:
other: more than 15% mean viability
Irritant / corrosive response data:
The mean viability of DOWANOL™ TPnB-H Glycol Ether was more than 50% at three minutes and more than 15% at one hour, hence based on the EpiDerm prediction model for corrosion, DOWANOL™ TPnB-H Glycol Ether was classified as non-corrosive.

Any other information on results incl. tables

Mean tissue viability of DOWANOL™ TPnB-H Glycol Ether treatment following the three minute exposure period was 89.2% and following the one hour exposure period was 92.6%. The test material, DOWANOL™ TPnB-H Glycol Ether showed a slightly higher viability value at one hour exposure time. This result may be due to the test compounds’ interaction with MTT reagent and difficulty to remove them from cell surface after one hour incubation. The pH value of the neat test material was measured using pH strip and the value was between 8.0 to 8.5. The pH value of 50% solution of TPnB-H in water was measured with pH meter as 12.7. The results of killed control test indicated a little or no test substance remained in the cell after removing the test substance and washing the cell disc follow manufacture’s protocol. The OD570 values were 0.246 and 0.295 for water treated killed-disc and TPnBH treated killed-disc, respectively. The mean raw absorbance of negative control (water) and test substance were then corrected for the amount of MTT reduced directly by both test material residues to obtain a final corrected OD values. The mean raw absorbance of negative control (water) and test substance (TPnB-H) were corrected for the amount of MTT reduced directly by both test material residues to obtain a final corrected OD570 values. The % of control viabilities were calculated base on the final corrected OD570 values. The mean viability of DOWANOL™ TPnB-H Glycol Ether was more than 50% at three minutes and more than 15% at one hour, hence based on the EpiDerm prediction model for corrosion, DOWANOL™ TPnB-H Glycol Ether was classified as non-corrosive.

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The mean tissue viability of DOWANOL™ TPnB-H Glycol Ether treatment following the three minute and one hour exposure periods were 89.2% and 92.6% respectively. Therefore, using the EpiDerm prediction model for corrosion DOWANOL™ TPnB-H Glycol Ether was classified as non-corrosive.
Executive summary:

DOWANOL™ TPnB-H Glycol Ether was evaluated for skin corrosivity potential in an EpiDerm In Vitro corrosion assay which utilizes cultured human epidermal cells. In this assay, test material was topically applied to a three-dimensional regenerated human epidermis tissue for three minutes or one hour. Skin corrosion was then expressed as the remaining cell viability after exposure to the test substance. The tissue cell viability was measured using a standard cytotoxicity assay MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide) and reported as a percentage of the mean of negative control. Skin corrosion potential of the test substance was classified according to tissue viability following exposure. A test substance is considered corrosive if the mean viability is less than 50% after three minutes or ≥ 50% at three minutes but less than 15% after one hour. Water served as a negative control and 8N potassium hydroxide was used as a positive control. The mean tissue viability of DOWANOL™ TPnB-H Glycol Ether treatment following the three minute and one hour exposure periods were 89.2% and 92.6% respectively. Therefore, using the EpiDerm prediction model for corrosion DOWANOL™ TPnB-H Glycol Ether was classified as non-corrosive.