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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-11-22 - 2014-05-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: MatTek Corporation; EpiOcular TM human cell construct 2010
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N-dimethyldodecanamide
EC Number:
221-117-5
EC Name:
N,N-dimethyldodecanamide
Cas Number:
3007-53-2
Molecular formula:
C14H29NO
IUPAC Name:
N,N-dimethyldodecanamide
Details on test material:
- Name of test material (as cited in study report): N,N Dimethyldodecane-1-amide
- Test-substance No.: 13/0555-1
- Physical state: liquid
- Analytical purity: N,N-Dimethyldodecanamide: 95.9 area-%; dodecanoic acid (lauric acid): 2.26 area-%; (for details see project No. AU134560-1)
- Lot/batch No.: 0009565072
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor.
- Homogeneity: The test substance was homogeneous by visual inspection.

Test animals / tissue source

Species:
other: in vitro test

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): EpiOcular: Fifty microliter (50 μL) of the undiluted liquid test substance was applied covering the whole tissue surface.

Duration of treatment / exposure:
After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed.
Observation period (in vivo):
not applicable (in vitro test)
Number of animals or in vitro replicates:
not applicable (in vitro test);
Details on study design:
Direct MTT reduction
To assess the ability of the test material to directly reduce MTT a pretest was performed as described below. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (de-ionized water) was tested concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results. In case that direct MTT reduction occurred, two freeze-killed control tissues were treated with, each, the test article and the negative control, in the same way as described in section “Experimental procedure” (3.6), additionally.
Basic procedure
Two tissues were treated with the test substance, the PC and NC, respectively. In addition two killed tissues were used for the test substance and NC, respectively, in order to detect direct MTT reduction. There are two separate protocols for liquids and solids, differing in exposure time and postincubation period. Due to the physical condition of the test substance the protocol for liquids was applied.
Pre-incubation of the tissues
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at standard culture conditions for 16 – 24 hours (pre-incubation).
Pretreatment of the tissues
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.
Application of the test substance
Using a pipette, fifty microliter (50 μL) of the undiluted liquid test substance was applied covering the whole tissue surface. Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC) or test substance (killed tissue control). After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed.
Removal of the test substance and postincubation period
To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium (post-soak immersion) in order to remove residual test substance. After 12 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 2 hours (postincubation period).
MTT incubation
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
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 tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

Results and discussion

In vivo

Results
Irritation parameter:
other: mean viability of the test-substance treated tissues [%]
Score:
30
Max. score:
100
Reversibility:
other: not applicable
Remarks on result:
other: in vitro test EpiOcular
Irritant / corrosive response data:
IVIS NC: 100% viability of NC
IVIS PS: 26% viability of NC

Any other information on results incl. tables

Findings of the EpiOcular Test

Individual and mean OD570 values, individual and mean viability values and inter-tissue variability


  1. Test substance


    tissue 1


    tissue 2


    mean KC

    inter-tissue

    mean and variability [%]


    NC

    mean OD570

    1.633

    1.533

    0.025

    1.583

    viability [% of NC]

    103.2

    96.8

    -

    100

    6.3


    13/0555-1

    mean OD570

    0.453

    0.483

    0.031

    0.468

    viability [% of NC]

    28.6

    30.5

    -

    30

    1.9


    PC

    mean OD570

    0.400

    0.416

    -

    0.408

    viability [% of NC]

    25.3

    26.3

    -

    26

    1.0


    Due to the ability of the test substance to reduce MTT directly, a KC was applied in parallel. However, the result of the KC did not indicate an increased MTT reduction (difference to KC of NC is not greater than 0.1; see section3.6.8). Thus the KC was not used for viability calculation.



Decision criteria for the combined assessment of BCOP and EpiOcular result (see also EpiOcular Test)

 BCOP result  EpiOcular result  Evaluation Test Strategy
 IVIS > 55 or HIS = IV  ≤ 60% viability  ocular corrosive or severe irritant
 IVIS < 55 and HIS < IV  ≤ 60% viability  irritant
 IVIS < 55 and HIS < IV  > 60% viability  Non-irritant

Applicant's summary and conclusion

Interpretation of results:
other: ocular corrosion or severe irritation in the in vitro eye irritation test strategy (combined result of BCOP and EpiOcular)
Conclusions:
Based on the results for BCOP and EpiOcular Test and applying the evaluation criteria N,N Dimethyldodecane-1-amide causes ocular irritation in the in vitro eye irritation test strategy under the test conditions chosen.
Executive summary:

EpiOcular

The potential of N,N Dimethyldodecane-1-amide to cause ocular irritation was assessed by a single topical application of 50 μL of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™).

Two EpiOcular™ tissue samples were incubated with the test substance for 30 minutes followed by a 2-hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The EpiOcular™ eye irritation test showed the following results:

The test substance is able to reduce MTT directly. However, this ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing. The mean viability of the test-substance treated tissues was 30%. Based on the observed results it was concluded, that N,N Dimethyldodecane-1-amide shows an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.

The result of the single EpiOcular does not exclude an severe irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study would be needed. Therefore a BCOP test was performed to receive additional information about the irritancy of the substance. Both results were used as a combined testing strategy (see endpointsummary for combined explaination).