Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
other: newly approved in vitro method
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21. June 2010 - 05. July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study under GLP, according to international guidelines and with full documentation

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:
other: Commission Regulation (EC) No. 761/2009, Method B.46: “In Vitro Skin Irritation: Reconstructed human epidermis model test” adopted 23. July 2009
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, Version 7.6, 9 September 2009
Deviations:
no
Qualifier:
according to
Guideline:
other: ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test (Altern Lab Anim. 2007 Dec; 35 (6): 559-601)
Deviations:
no
Qualifier:
according to
Guideline:
other: Protocol for IN VITRO EpiDermTM SKIN IRRITATION TEST (EPI-200-SIT), Rev. 3/23/2009, MatTek Corporation, Ashland, MA 01721, USA
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- The analysis was performed for the reference sample "Sample 2010" as defined in Section 1.4
- Lot/batch No.: 10013593
- Expiration date of the lot/batch: March 2011
- Storage condition of test material: For several days, the test item was stored in the test facility at room temperature protected from light and kept under inert gas. As soon as a fridge with explosion prevention was available, the test item was stored at 2 – 8 °C. No relevant changes in composition were detected before and after storage at room temperature (GC chromatograms).

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
human-derived epidermal keratinocytes (EPI-200 tissues)
Justification for test system used:
OECD approved and validated test system, suitable to replace in vivo studies
Vehicle:
unchanged (no vehicle)
Details on test system:
The EpiDermTM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cultures inserts.
Origin: Epi-200 tissues were procured from MatTek Corporation in Ashland, USA. Day of delivery: 1. July 2010 batch: 13657
pre-incubation of tissues at 37 °C and 5% CO2 for one hour; thereafter with fresh assay medium (0.9 mL) at 37 °C and 5% CO2 for 18 hours
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Applied volume: 30 µL of test item
Duration of treatment / exposure:
35 - 60 minutes in incubator and then 24 hour incubation
Duration of post-treatment incubation (if applicable):
18 ±2 hours
Number of replicates:
One plate (three tissues) as negative control, one plate (three tissues) as positive control, one plate was used for the test item

Test system

Type of coverage:
other: nylon mesh
Amount / concentration applied:
See "Any other information on materials and methods incl. tables".
Duration of treatment / exposure:
See "Any other information on materials and methods incl. tables".
Observation period:
See "Any other information on materials and methods incl. tables".
Number of animals:
Not applicable

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
Formazon production
Run / experiment:
mean of three tissues
Value:
56.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Absorption values for negative control, test item and positive control (mean of 2 measurements, blank value substracted)

Designation

Negative Control

Test Item

Positive Control

Mean – blank (Tissue 1)

2.389

1.348

0.171

Mean – blank (Tissue 2)

2.244

1.240

0.182

Mean – blank (Tissue 3) 

2.129

1.244

0.177

Mean of the three Tissues

2.254

1.277

0.177

Relative Standard Deviation
of the three tissues

5.8%

4.8%

3.1%

Table 2: % Formazan production for the test item and positive control

Designation

Test Item

Positive Control

% Formazan production (Tissue 1)

59.8%

7.6%

% Formazan production (Tissue 2)

55.0%

8.1%

% Formazan production (Tissue 3)

55.2%

7.9%

% Formazan production Mean

56.7%

7.8%

Assessment

The relative absorbance values were reduced to 56.7% after the treatment. This value is above the threshold for irritation (50%). Therefore, the test item is considered as not irritant.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is considered not irritant. After the treatment, the relative absorbance values were decreased to 56.7%. This value is above the threshold for irritation (50%).
The optical density of the negative control was well within the required acceptability criterion of 1.0 < mean OD < 2.5. The positive control induced a decrease in the relative absorbance as compared to the negative control to 7.8 % (required: <= 20%) thus ensuring the validity of the test system. Variation within replicates was within the accepted range for negative control, positive control and test item. For these reasons, the result of the test is considered valid.
Executive summary:

The skin irritation potential of „2-propanol and 2-butanol production, distn. residue“ in theHuman Skin Model Test was determined following EU-Method B.46. One valid experiment under GLP was performed. Three tissues of the human skin model EpiDerm(TM) were treated with „2-propanol and 2-butanol production, distn. residue“ for 60 minutes.

30 µL of the liquid test item (using a nylon mesh) were applied to each tissue and spread to match the tissue size. DPBS-buffer was used as negative control, 5% SDS-solution was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 1.0 < mean OD < 2.5. The positive control showed clear irritating effects. Variation within tissues was acceptable (< 18%).

After the treatment with the test item, the relative absorbance values were reduced to 56.7%. This value is above the threshold for irritation potential (50%). Therefore, „2-propanol and 2-butanol production, distn. residue“ is considered as not irritant in the Human Skin Model Test.