Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
other: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Justification for read-across is given in Section 13 of IUCLID.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The study was performed according to the methods described in the following publications:
MatTek Corporation, Ashland, MA 01721, USA: EpiOcularTM human cell construct: Procedure details, Version 3.1a of February 10, 2010 and
Harbell J.W. et al. (2009): COLIPA Program on Optimization of Existing in-vitro Eye Irritation Assays for Entry into Formal Validation: Technology Transfer and Intra/Inter Laboratory Evaluation of EpiOcular Assay for Chemicals. Poster # 378, Society of Toxicology, March 2009.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Didodecyl fumarate
- Physical state: solid
- Analytical purity: 93.8 area-%
- Lot/batch No.: 0008043725

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
TEST EYE MODEL
- Description of the cell system used: EpiOcularTM; reconstructed three-dimensional human cornea model (OCL-200). The model represents a reconstructed three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratiozytes used to model the human corneal epithelium. Irritant materials are identified by their ability to induce cytotoxicity (= loss of viability) at the surface of the EpiOcularTM tissue. The decrease in cell viability is determined by MTT reduction assay.
- Source: MatTek Corporation, Ashland MA, USA.

ADAPTATION TO CELL CULTURE CONDITIONS: upon receipt, tissues were transferred into 6-well plates containing 1 ml assay medium per well and preincubated in a humidified incubator for 16 to 24 hours (37 ± 1 °C, CO2) before use. After the pre-incubation the tissues were pre-treated with 20 µl of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.

INCUBATION CONDITIONS
- Temperature: 37 ± 1 °C
- CO2 gas concentration: 5 %
- Humidity: 90 - 95 %

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 µl bulk volume (about 22 mg)

NEGATIVE CONTROL:
- 50 µl of sterile de-ionized water.

POSITIVE CONTROL SUBSTANCE:
- Amount applied: 50 µl.
Duration of treatment / exposure:
90 minutes
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
the test was performed in duplicates for each treatment and control group.
Details on study design:
TEST SITE
- Area of exposure: 0.6 cm².

REMOVAL OF TEST SUBSTANCE
- Washing: the test item was washed from the tissue three times with phosphate buffered saline (PBS). In order to remove residual test substance, washed tissues were immediatly immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium (post-soak immersion). After 12 minutes of post-soak immersion, each tissue was dried and transferred to fresh 6-well plates filled with pre-warmed medium.
- Time after start of exposure: 90 min.

CELL VIABILITY MEASUREMENTS: for determining alterations in cell viability, MTT reduction assays were performed 18 h after the incubation period. Therefore, tissues were incubated in 300 µl MTT solution for 3 h at 37 ± 1 °C and 5 % CO2. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissue in isopropanol. The optical density was measured at 570 nm wavelength in a plate spectrophotometer.

ACCEPTANCE CRITERIA
Negative control (NC): the absolute OD570 of the negative control tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.
Positive control (PC): methyl acetate used as PC usually leads to a tissue viability of approx. 25 %. A viability of < 50 % is acceptable.
Tissue variability: two tissues were treated under the same conditions. A variability between the tissues is considered to be acceptable if the difference of the viability is ≤ 20 %.
 
EVALUATION CRITERIA
A chemical is considered as irritant when the tissue viability was less than or equal to 50 % after the exposure and post-treatment incubation period. At present no prediction is performed if the mean relative tissue viability with a test material is > 50 % ≤ 60 % as the cut off value is currently being evaluated to lie in this range. If the tissue viability after exposure and post-treatment incubation period is above 60 %, the test chemical may be considered as non-irritant.

Results and discussion

In vitro

Results
Irritation parameter:
other: cell viability %
Run / experiment:
90 min
Value:
82
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean value of three replicates

Any other information on results incl. tables

Table: cell viability results

test substance

 

tissue 1

tissue 2

mean

SD

NC

mean OD570

1.646

1.393

1.52

 

viability

[% of NC]

108.3

91.7

100

16.6

test item

mean OD570

1.133

1.349

1.241

 

viability

[% of NC]

74.6

88.7

82

14.2

PC

mean OD570

0.43

0.344

0.387

 

viability

[% of NC]

28.3

22.6

25

5.7

Applicant's summary and conclusion

Interpretation of results:
other: not classified as an eye irritant according to the CLP Regulation (EC) No.1272/2008
Conclusions:
Non irritating to eye; cell viability = 82 %
Executive summary:

The eye irritation potential of the category substance was assessed in the in-vitro study by using the EpiOcular model. The test chemical was applied topically to three tissues of a reconstructed three-dimensional human cornea model and the tissue viability was measured following exposure of 90 min and a post-treatment incubation period of 18 hours. The decrease in cell viability was determined by MTT reduction assay. The tissues, after the removal of the substance, were incubated with MTT medium and the optical density of the extracted formazan was measured at 570 nm. Negative and positive controls run in parallel; the % photometric absorption of test item and positive control was calculated by comparison with the negative control.

Irritant materials are identified by their ability to induce cytotoxicity (= loss of viability) at the surface of the EpiOcular tissue.

Cell viability of the tissues treated with the test substance was 82 % (mean value of three replicates). These values are laying above the threshold for irritation.

The category member substance is not considered to be irritating to the eye.