Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 July 2014 - 14 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Remarks:
Epi-200 SIT
Source species:
human
Cell type:
other: epidermal keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200- SIT
- Tissue batch number(s): 19652 Kit G
- Date of initiation of testing: 09 July 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were carefully dried using sterile cotton tipped swap.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
On the day of testing the MTT concentrate was diluted with the MTT diluent. The 24-well plates were prepared before the end of the tissue pre-warming period. A volume of 300 µL of the MTT solution was added to each well and the plates were kept in an incubator (37 ± 1 °C, 5 ± 0.5 % CO2) until further use.
After the 42-hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT-plates. After a 3-hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2), the MTT solution was aspirated from the wells, and the wells were rinsed three times with DPBS. Inserts were transferred onto new 24-well plates. The inserts were immersed into extractant solution by gently pipetting 2 mL extractant solution (isopropanol) in each insert. The level rose above the upper edge of the insert, thus tissues were completely covered from both sides. The 24-well plate was sealed to inhibit the isopropanol evaporation. The formazan salt was extracted for 69 hours at room temperature.
After the extraction period was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 x 200 µL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. OD was read in a microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1) with a 570 ± 1 nm filter. Mean values were calculated from the 3 wells per tissue.


PREDICTION MODEL / DECISION CRITERIA
For the current test, an irritation potential of a test item according to EU classification R38 (according to directive 67/548/EEC), H315 (according to regulation (EC) 1272/2008), and GHS category 2 according to UN GHS (published 2003, last (3rd) revision 2009) is recommended if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control.
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):30 µL of the undiluted test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS was used as negative control.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5% SLS solution in deionised water was used as positive control.
Duration of treatment / exposure:
After pre-incubation of EpiDerm™ tissues was completed, medium was replaced by 0.9 mL of fresh medium per well. The negative and positive control and the test item were added into the insert atop the corresponding EpiDerm™ triplicate tissues. The 6-well plates were placed into the incubator for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2, 95 ± 5% RH. Thereafter, plates were removed from the incubator and placed into the sterile hood until the period of 60 minutes was completed.
Number of replicates:
3

Test animals

Species:
other: human keratinocytes

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
30 µl of the undiluted test item was dispensed directly atop the EpiDerm™ tissue.
Duration of treatment / exposure:
60 minutes
Details on study design:
This in vitro study was performed to assess the irritation potential of Di-(iso)-pentyl terephthalate (DPT) by means of the Human Skin Model Test.
Three tissues of the human skin model EpiDerm™ SIT (EPI-200) (from MatTek Corporation, Bratislava, Slovakia) were treated with the test item, the negative or the positive control for 60 minutes.Each 30 µL of the test item, of the negative control (DPBS), and of the positive control (5% SLS) were applied to each of triplicate tissue and spread to match the tissue size.
The test is followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential and is used for the purpose of classification as irritating or non-irritating.

The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated.

For the test item and the positive control the mean relative viability +/- rel. standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model:

in vitro result in vivo prediction
mean tissue viability < 50% irritant (I), H315 (category 2)
mean tissue viability > 50% non-irritant (NI)

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean relative absorbance value of the test item is corresponding to the cell viability.
Run / experiment:
DPT
Value:
80.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Any other information on results incl. tables

Results after treatment with Di-(iso)-pentyl terephthalate (DPT) and the controls

Dose Group

Treat-ment Interval

Absor-bance 570 nm
Tissue 1*

Absor-bance 570 nm
Tissue 2*

Absor-bance 570 nm
Tissue 3*

Mean Absor-bance of 3 Tissues

Rel. Absor-bance [%] Tissue 1, 2 + 3**

Relative Standard Deviation

[%]

Mean Rel. Absorbance

[% of Negative Control]***

Nega-tive Control

60 min

1.950

2.003

2.336

2.096

93.0
95.5
111.4

10.0

100.0

Positive Control

60 min

0.096

0.104

0.112

0.104

4.6
5.0
5.2

7.5

5.0

Test Item

60 min

1.608

1.640

1.790

1.680

76.7
78.2
85.4

5.8

80.1

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 80.1% (threshold for irritancy:≤50%), consequently the test item was not irritant to skin. 

 

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
In conclusion, the test item Di-(iso)-pentyl terephthalate (DPT) is not irritant to skin according to UN GHS and EU CLP regulation in this study.