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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:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test substance : 2,2'-oxydiethanol
- Test-substance No.: 07/0335-4
- Batch identification : OOOSTD77LO
- CAS No.:111-46-6
- Purity: min. 99.85 % (analytical results given by the sponsor per mail)
- An analytical characterization of the test substance according to GLP has been performed.
- ldentity: Confirmed
- Homogeneity: The test substance is homogeneous by visual inspection.
- Storage stability: The sponsor guarantees the storage stability at least until 08 Oct 2016.
- pH-value: Ca. 5 (undiluted test substance; determined in the lab prior to start of the GLP study)
- Physical state I color: Liquid I colorless clear
- Storage conditions : Room temperature, avoid temperatures > 40 °C; under light exclusion, protected against humidity.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: Three dimensional human epidermis model
Vehicle:
physiological saline
Details on test system:
THREE DIMENSIONAL HUMAN EPIDERMIS MODEL
The EpiDermTM model cinsists of normal, human-derived epidermal keratinocytes which have been cultured for a multilayered, highly differentiated model of the human epidermis. It sonsists of organized basal, spinous and granular layers, and multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous tothose found in vivo. The EpiDerm TM tissues (surface 0.6 cm2) are cultured on cellular inserts (MILLICELLS R, 10mm2) and are commercially available as kits (EpiDermTM200), containing 24 tissues on shipping agarose.

The test was performed under sterile conditions working at a laminar flow hood. All materials used were sterilized if possible or disinfected with 70% isopropanol.

1. PREINCUBATION OF THE TISSUES
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 ml assay medium and preconditioned in the incubator at 37°C. After 1 hour the pre-incubation medium was replaced with fresh medium and preconditioning continues for 18 ± 3 hours.

2. APPLICATION OF THE TEST SUBSTANCE
Three tissues were treated with each test substance , the PC and NC, respectively. Three additional tissues (KC) were used for the test substance and the NC, respectively; if a positive reaction has been observed in the MTT reduction test described under 5.
Thirty microliters (30 µL) of the undiluted liquid test substance were applied using a pipette. lf applicable a nylon mesh was placed carefully onto the tissue surface afterwards.
Control tissues were concurrently treated with 30 µL sterile PBS (NC, NC KC (if applicable)) or 5% SOS (PC) or test substance (KC, if applicable). After application a nylon mesh was placed carefully onto each tissue surface of the NC and PC and if applicable NC KC and test substance KC.
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator .
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab.
Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours.

3. MEDIUM CHANGE
After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.

4. MTT incubation
After the postincubation period,the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation.

5. DETECTION OF MTT METABOLISM
The formazan that is metabolically produced by the tissues was extracted by incubation of the tissues in 2 mL isopropanol for at least 2 hours at room temperature on a plate shaker (ca. 120 rpm). After shaking the isopropanol extract and piercing the tissues, 2 aliquots of each extract per tissue were transferred to a 96-well microtiter plate. The optical density (00510) was determined spectrophotometrically using a filter with a wavelength of 570 nm.
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
30 µL per well
Duration of treatment / exposure:
24 ± 2 hours
Duration of post-treatment incubation (if applicable):
18 ± 2 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
94.5
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

 

Test substance identification

 

tissue

1

tissue

2

tissue 3

mean

SD

CV [%]

NC

mean OD570

2,330

2,267

2,599

2,399

 

 

viability
[% of NC]

97,1

94,5

108,3

100,0

7,3

7,3

07/0335-4

mean OD570

2,696

2,000

2,107

2,268

 

 

viability
[% of NC]

112,4

83,4

87,8

94,5

15,6

16,5

PC

mean OD570

0,074

0,072

0,072

0,072

 

 

viability
[% of NC]

3,1

3,0

3,0

3,0

0,0

1,6

NC = negative control

PC = positive control

07/0335-4 = test substance 2,2'-oxydiethanol

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met