Registration Dossier

Administrative data

Endpoint:
skin irritation / corrosion, other
Remarks:
EPISKIN
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7th June 2010 - 14th June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Principles of method if other than guideline:
OECD Guideline For The Testing of Chemicals (2009), Draft proposal for a new guideline: In Vitro Skin Irritation: Human Skin Model Test. Year 2009. EPISKIN three dimensional human skin model.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Diisopentyl phthalate
EC Number:
210-088-4
EC Name:
Diisopentyl phthalate
Cas Number:
605-50-5
Molecular formula:
C18H26O4
IUPAC Name:
diisopentyl phthalate
Details on test material:
- Name of test material (as cited in study report): Diisoamyl phthalate
- Physical state: colourless to slightly yellow clear liquid
- Analytical purity: 99.98%
- Purity test date: Not reported
- Lot/batch No.: 7920-1-1
- Expiration date of the lot/batch: 12 August 2012
- Storage condition of test material: Ca. room temperature (+20°C) in the dark

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:The EPISKIN Skin Irritation Test-42 hours
- Date of initiation of testing: 8th June 2010

TEMPERATURE USED FOR TEST SYSTEM
The tissues were removed from the agar and placed into wells of 12 well plates containing
2 ml pre-warmed maintenance medium per well. The tissues were incubated for a minimum
of 24 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.

REMOVAL OF TEST MATERIAL AND CONTROLS
After 15 ± 0.5 minutes, each tissue was rinsed with 25 ml sterile Dulbeccos Phosphate
Buffered Saline (DPBS) to remove residual test substance.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
It is possible that a test substance may reduce MTT, resulting in the production of a
blue/purple colour without any involvement of cellular mitochondrial dehydrogenase. Since
the test substance is rinsed off the tissue before the MTT assay, this is usually avoided.
However, it is possible that small amounts of test substance may be present after washing or
be released through the tissue into the MTT medium. If the mixture with the test substance
turned blue/purple after approximately 3 hour incubation at 37 ±2ºC in 5% CO2 in air, MTT
reduction may have occurred.
The MTT reducing capability of the test substance, Diisoamyl Phthalate, was investigated by
mixing approximately 10 Fl of the test substance with 2 ml of 0.3 mg/ml MTT solution in
duplicate. A control of 10 Fl of purified water, mixed with 2 ml of 0.3 mg/ml MTT solution
was also included in duplicate.

NUMBER OF REPLICATE TISSUES: Triplicate tissues.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
Control samples:
yes, concurrent positive control
Amount/concentration applied:
10 μl


Duration of treatment / exposure:
Triplicate tissues were dosed for 15 ± 0.5 minutes with the test substance.
Duration of post-treatment incubation (if applicable):
42 hours.
Number of replicates:
3

Test system

Type of coverage:
other:
Preparation of test site:
other: n/a
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): 100%
Duration of treatment / exposure:
15+/-0.5 minutes
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
See any other information below.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
104.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
74.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
104.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
94.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Negative control
The mean absorbance of the triplicate negative control values was 0.711 which was above the minimum acceptance value of 0.6. The standard deviation of the % viability was 13.8 which was below the maximum acceptance value of 18.

Positive control
The percentage mean viability of the positive control was 12.0 ± 1.7 of the negative control. These were below the maximum acceptance values of 30% viability and SD of 18.

Test substance
It was concluded that the test substance, Diisoamyl Phthalate, with a mean tissue viability of 94.6 ± 17.3%, was predicted as non-irritant to the skin.

In vivo

Irritant / corrosive response data:
Not Applicable

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
It was concluded that the test substance, diisopentyl phthalate, with a mean tissue viability of 94.6 ± 17.3%, was predicted as a non-irritant to the skin.
Executive summary:

The objective of this test was to assess the skin irritation potential, in vitro, of the test ubstance, Diisoamyl Phthalate.

The test substance was applied to EPISKIN human epidermis skin constructs. The constructs consisted of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis with a functional multilayered stratum corneum. The cell viability of the multi layers was determined by mitochondrial dehydrogenase activity. The prediction model uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances.

The test substance, Diisoamyl Phthalate, elicited a mean tissue viability of 94.6% and was predicted as non-irritant.