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Administrative data

Description of key information

One in vitro skin irritation study and one in vitro eye irritation/corrosion study were conducted. In all studies no classification of skin and eye irritation were needed.  

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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
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
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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11.01.2016 to 16.03.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: Freshly isolated bovine cornea (at least 9 month old donor cattle)
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: abattoir: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Characteristics of donor animals (e.g. age, sex, weight): 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The isolated eyes were
transported to the laboratory in HBSS at ambient temperature.
- Time interval prior to initiating testing: The corneae were directly used in the BCOP test on
the same day.

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL


Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
two hours
Number of animals or in vitro replicates:
3 replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such
as vascularization, pigmentation, opacity and scratches were discarded. The cornea was
carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of
tissue (sclera) was left for stability and handling of the isolated cornea.


NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
Saline (0.9% NaCl in deionised water)

POSITIVE CONTROL USED
2-Ethoxyethanol (purity: 99%)

APPLICATION DOSE AND EXPOSURE TIME
The test item were applied undiluted for ten minutes.

TREATMENT METHOD:
Each isolated cornea was mounted in a specially designed cornea holder according to the
description given in OECD guideline 437, that consists of anterior and posterior
compartments, which interface with the epithelial and endothelial sides of the cornea,
respectively. The endothelial side of the cornea was positioned against the sealing ring (Oring)
of the posterior part of the holder. The cornea was gently flattened over the O-ring but
stretching was avoided. The anterior part of the holder was positioned on top of the cornea
and fixed in place with screws. Both compartments of the holder were filled with incubation
medium. The posterior compartment was filled first to return the cornea to its natural convex
position.

POST-INCUBATION PERIOD: no


REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: one
- POST-EXPOSURE INCUBATION: two hours

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacitometer determines changes in the light transmission passing through the corneae,
and displays a numerical opacity value. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the
manual and the opacity of each of the corneae was determined by reading each holder placed
in the photoreceptor compartment for treated cornea.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = opacity value + (15 x OD490 value)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the
current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less
than the established upper limits for background opacity and permeability values for
bovine corneae treated with the respective negative control.
Irritation parameter:
cornea opacity score
Value:
ca. 0
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
GHS criteria not met
Conclusions:
Relative to the negative control, the test item DINCD (Diisononyl 1,4-
cyclohexanedicarboxylate) did not cause any increase of the corneal opacity or permeability.
The calculated mean in vitro irritancy score was 0.00.
In conclusion, according to the current study and under the experimental conditions reported,
DINCD (Diisononyl 1,4-cyclohexanedicarboxylate) is not categorized (GHS).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro skin irritation test:

This in vitro study was performed to assess the irritation potential of DINCD (Diisononyl

1,4-cyclohexanedicarboxylate) by means of the Human Skin Model Test according to OECD

TG 439.

Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative

or the positive control for 15 minutes (26.3 μL/cm2).

After treatment with the test item the mean relative absorbance value did not decrease

(109.3%) compared with the viability value after exposure to the negative control. This value

does not affect the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered

to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions

reported, the DINCD (Diisononyl 1,4-cyclohexanedicarboxylate) is not irritant to skin

according to UN GHS and EU CLP regulation.

Bovine corneal opacity and permeability assay (BCOP):

This in vitro study was performed to assess the corneal damage potential of DINCD

(Diisononyl 1,4-cyclohexanedicarboxylate) by means of the BCOP assay using fresh bovine

corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the

positive and the negative controls were applied to corneae fixed in an incubation chamber in

horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation

medium. After the incubation phase the test item, the positive and the negative controls were

each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at

32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as

well. Afterwards, opacity was measured a second time (t130).

After the opacity measurements permeability of the corneae was determined by measuring

spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal

position for 90 minutes at 32 ± 1 °C.

Relative to the negative control, the test item DINCD (Diisononyl 1,4-

cyclohexanedicarboxylate) did not cause any increase of the corneal opacity or permeability.

The calculated mean in vitro irritancy score was 0.00. According to OECD 437 (see table in

chapter 3.8.3) the test item is not categorized (GHS).

Justification for classification or non-classification

Based on the available studies, the test substance is not classified for skin or eye irritation according to EC Regulation No. 1272/2008.