Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A study according to the EU and OECD methods, including GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
Only 2 tissues could be used for the test substance; without affecting the validity of the test.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
Only the draft of the guideline 431 was available in 2009.
Deviations:
yes
Remarks:
Only 2 tissues could be used for the test substance; without affecting the validity of the test.
Principles of method if other than guideline:
The test consists of topical exposure of the neat test substance to a human reconstituted epidermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT, present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of viability of tissue exposed to chemicals in comparison to negative controls is used to predict skin irritation potential.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Common name: sodium phenoxyacetate
CAS No: 3598-16-1
Purity (dry basis): 99.83 %
Appearance: off white powder
Conditions of storage: Room temperature, no light protection
Stability at conditions of storage: Stable
Batch No: 30031722 or WE 30031722
Date of expiry: 13 January 2011
Supplier: Sandoz GmbH

Test animals

Species:
other: In vitro test using human reconstituted epidermis.

Test system

Type of coverage:
open
Preparation of test site:
other: none
Vehicle:
other: Phosphate buffered saline (PBS)
Amount / concentration applied:
The application spoon was filled with 25 mg finely grounded test substance. The "spoonful" was levelled by gently scratching the excess material away, avoiding compression. 25 µL PBS were added for wetting of the tissue.
30 µL of each reference substance.
Duration of treatment / exposure:
1 hour.
Observation period:
MTT-test after a post-exposure period of 42 hours.
Number of animals:
3 tissue replicates were used.
Details on study design:
MatTek's EpiDerm System consists of normal, human-derived epidermal keratinocytes which have been cultured form a multilayered, 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 EpiDerm™ tissues (surface 0.6 cm2) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm Ø) and shipped as kits, containing 24 tissues on shipping agarose.

Negative control: distilled water.
Positive control: 5 % sodium dodecylsulfate (SDS).

MTT-test:
After incubation with the test substance, post-incubation and washing with PBS, the tissues were incubated with MTT medium at 37 °C and 5 % CO2. After 3 hours, the MTT medium was aspirated from all wells and the tissues were gently rinsed with PBS (2 times). For extraction, the tissues were incubated with extractant solution (isopropanol) for 2 hours with shaking.
After the extraction period, the tissues were pierced with an injection needle and the extract (now a blue formazan solution) was allowed to run into the well from which the tissue was taken. The 24-well plates were placed on a shaker for 15 minutes until the solutions were homogeneous in colour.

Cell viability measurement:
Per each tissue 2 x 200 µL aliquots of the blue formazan solution were transferred into a 96- well flat bottom microliter plate and the optical density (OD) was measured using the extractant solution as blank in a plate spectrophotometer at 570 nm, without reference filter.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
69
Vehicle controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
87.7
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The tissue viabilities of 69.0 and 87.7 % (mean: 78.4 %) for the test substance indicate "no irritation".

Any other information on results incl. tables

Assay acceptance criteria:

• The mean OD 570 of the negative control tissues is≥1.0 and≤2.5. Actually obtained for water: 1.569.

• The mean tissue viability of the positive control is≤20 %. Actually obtained for 5 %-SDS: 10.5 %.

• The standard deviation calculated from individual percentual tissue viabilities of the 3 identically treated replicates is <18. Actually obtained for the test substance treated tissues: 13.2.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
Under the experimental conditions of this study, the test substance is considered to be 'non-irritant' to skin.
Executive summary:

The EpiDerm Skin Irritation Test (Model EPI-200) was performed to reveal possible irreversible tissue damages of the skin following the application of sodium phenoxyacetate. The test substance was topically applied for 60 minutes to the epidermal surfaces of three­ dimensional human epidermis models. After a post-incubation of 42 hours, a cell viability test was performed.

Investigations performed were in conformance with the EC-method B.46 "In vitro skin irritation: Reconstructed Human Epidermis Model Test" and the draft OECD Guideline "In vitro Skin irritation: Reconstructed Human Epidermis (RhE) Test Method".

The mean percentage viability of the treated skin discs was 78.4 % which is above the threshold of 50 % for classification. According to the results of this study and the Directive 2001/59/EC, sodium phenoxyacetate is considered to be non-irritant to skin.