Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27.01.2016 to 18.04.2016
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vitro test system

Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin™
- Tissue batch number(s): 16-EKIN-009
- Delivery date: 01 March 2016
- Date of initiation of testing: 01 March 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Number of washing steps: one


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT per well
- Incubation time: 3 hours
- Spectrophotometer: microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1)
- Filter :570 ± 1 nm


NUMBER OF REPLICATE TISSUES: Triplicates

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
For correct interpretation of results it is necessary to assess the ability of the test item to
directly reduce MTT. To test for this ability 10 μL of the test item was added to 2 mL of
MTT solution (0.3 mg/mL) and the mixture was incubated in the dark at
37 ± 1.5 °C (5 ± 0.5% CO2) for 3 hours. MTT-solution containing 10 μL of DMEM was used
as control.
Since the colour did not turn blue/purple, the test item was not considered to be a MTT
reducer. Therefore, an additional test with freeze-killed tissues was not necessary to be
performed.


PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the mean tissue viability after 15 minutes exposure is less or equal than 50%.
- The test substance is considered to be non-irritant to skin if the mean tissue viability after 15 minutes exposure is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 μL



NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): 5%
Duration of treatment / exposure:
15 minutes
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
ca. 109
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
After treatment with the test item the mean relative absorbance value (109.3%; threshold for
irritancy: ≤ 50%) was not reduced compared to the viability resulted after exposure to the
negative control.
In conclusion, it can be stated that in this study and under the experimental conditions
reported, DINCD (Diisononyl 1,4-cyclohexanedicarboxylate) is not irritant to skin
according to UN GHS and EU CLP regulation.