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Administrative data

Description of key information

Based on the results of an in vitro skin irritation test conducted according to OECD 439 guideline and GLP principles, it is concluded that dibutyl itaconate is not irritating in the in vitro skin irritation test. Furthermore, a BCOP assay was conducted in accordance with OECD/EC guidance and GLP principles, based on which it is concluded that DBI does not have irritant or corrosive effects on the eye.

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:
08DEC2015 - 11JAN2016
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)
Version / remarks:
(adopted 28 July 2015)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
(20 July 2012)
Deviations:
no
GLP compliance:
yes
Species:
human
Details on test animals and environmental conditions:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 36.3 - 37.2
- Humidity (%): 71-90
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 25 μl

NEGATIVE CONTOL:
- Amount applied: 25 µl phosphate buffered saline

POSITIVE CONTROL
- Amount applied: 25 µl
- Concentration: 5% (aq) sodium dodecyl sulphate
Duration of treatment / exposure:
15 minutes
Details on study design:
TEST SITE
- EPISKIN Small Model (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-001).This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days , which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
121
Vehicle controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
7%
Remarks on result:
no indication of irritation

The test substance was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that the test substance did not interact with MTT. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly.

Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that dibutyl itaconate is not irritating in the in vitro skin irritation test.
Executive summary:

In an in vitro skin irritation test using a human skin model (EPISKIN Small Model), the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2 cultured skin (25 μl). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 7% whereas the test substance showed cell viability of 120%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that dibutyl itaconate is not irritating in the in vitro skin irritation test.

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:
01DEC2015 - 04DEC2015
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)
Version / remarks:
(adopted July 26, 2013)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
(09 December 2010)
Deviations:
no
Qualifier:
according to
Guideline:
other: as summarized under "Principles of method if other than guideline"
Principles of method if other than guideline:
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) study plan 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
- Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.
GLP compliance:
yes
Species:
cattle
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: approx. 750 µL per cornea

NEGATIVE CONTROL
- Amount applied: 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount applied: 750 µl per cornea
Concentration (if solution): 20% (w/v) Imidazole


Duration of treatment / exposure:
10 ± 1 minutes
Duration of post- treatment incubation (in vitro):
After exposure the cornea is thoroughly rinsed to remove the test substance followed by incubation for 120 ± 10 minutes in cMEM. Then, corneas were incubated in sodium-fluorescein solution for 90 ± 5 minutes (both incubations at 32 ± 1°C).
Number of animals or in vitro replicates:
3
Details on study design:
TEST SITE
- Isolated bovine cornea

REMOVAL OF TEST SUBSTANCE
- Washing: yes (with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation) and thereafter with cMEM)
- Time after start of exposure: 10 minutes

SCORING SYSTEM:
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).


TOOL USED TO ASSESS SCORE:
- opacitymeter and microplate reader

DATA EVALUATION:
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces an IVIS > 55 is defined as a corrosive or severe irritan (UN GHS: catgeory 1);
For a test substance that induces an IVIS >3 and ≥ 55, no prediction on irritant potency can be made.

Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
-0.1
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks:
IVIS = 121
Remarks on result:
no indication of irritation
Interpretation of results:
GHS criteria not met
Conclusions:
Based on an BCOP assay conducted in accordance with OECD/EC guidance and GLP principles, DBI does not have irritant or corrosive effects on the eye.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In an in vitro skin irritation test using a human skin model (EPISKIN Small Model), the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2cultured skin (25 μl). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 7% whereas the test substance showed cell viability of 120%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that dibutyl itaconate is not irritating in the in vitro skin irritation test.

Based on an BCOP assay conducted in accordance with OECD/EC guidance and GLP principles, DBI does not have irritant or corrosive effects on the eye.

Justification for classification or non-classification

Based on the available data, DBI is not classified for skin irritating properties or eye irritating properties according to Regulation EC (No.) 1272/2008 (CLP).