Reaction mass of N,N-dimethyldodecanamide and N,N-dimethyltetradecanamide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07-01-2014 to 26-05-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

The BCOP test was conducted according to the following test guidelines:
- OECD Guideline for Testing of Chemicals No. 437, 26 July 2013 (“Bovine Corneal
Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye
Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye
Damage”)
- Commission Regulation (EU) No. 1152/2010 of 8 December 2010 amending for the
purpose of its adaptation to technical progress, Commission Regulation (EC) No
440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No
1907/2006 of the European Parliament and of the Council on the Registration, Evaluation,
Authorisation and Restriction of Chemicals (REACH), Part B: Methods for the
determination of toxicity and other health effects: Bovine Corneal Opacity and
Permeability Test, Method for identifying ocular corrosives and severe irritants; Official
Journal of the European Union, No. L 324

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

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

Specific details on test material used for the study:

Name of test substance: N,N Dimethyldodecane-1-amide
Test-substance No.: 13/0555-1
Batch identification: 0009565072
CAS No.: 3007-53-2

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Isolated bovine cornea:

The test system (target tissue) is the isolated bovine cornea.
Bovine eyes are obtained as a by-product of freshly
slaughtered cattle (age of the animals: minimum 12 months,
maximum 60 months).

Supplier:

Schlachthof Bensheim, Am Schlachthof 7-9, 64625 Bensheim,
Germany

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Single topical application of 750 μL of the undiluted test substance to the
epithelial surface of isolated bovine corneas.

Duration of treatment / exposure:

Three corneas were treated with the test substance for 10 minutes followed by a 2-hours
post-incubation period.

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3 corneas per dosage tested

Details on study design:

Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagles’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour. After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 530 opacity units1 were discarded. Corneas with opacity values close to the median value of all corneas were selected as negative control (NC). The remaining corneas were then distributed into treatment and positive control groups. Each corneal holder was uniquely identified with a number on the chambers.
Application of the test substance and washing:
Each treatment group (test substance, NC and PC) consisted of 3 corneas. Before application the medium in the anterior chamber was removed using a syringe. 750 μL of the undiluted liquid test substance was applied into the anterior chamber using a pipette. Control tissues were concurrently applied into the anterior chamber with 750 μL of de-ionized water (negative control, NC) or with 750 μL of 100% dimethylformamide (positive control, PC) using a pipette.
The corneas were incubated in a horizontal position at about 32 °C for approximately 10 minutes (liquids and surfactants). The test substance, NC and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).
The corneas were incubated for further 2 hours at about 32 °C. After the incubation period the medium was removed and both chambers were then refilled with fresh Eagle’s MEM.
Measurement of final corneal opacity:
Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
Determination of permeability:
For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (4 mg/mL for liquid test substances and surfactants) and incubated for 90 ± 5 min in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.
Histopathology:
After determination of opacity and permeability, the corneas will be fixed in 4% formaldehyde for at least 24h and transferred to the laboratory of General Pathology for further histotechnical processing and examination by light microscopy.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Opacity score of the test substance

Value:

ca. 11.1 - ca. 14.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Opacity score of the negative control

Value:

ca. 2.4 - ca. 6.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

Opacity score of the positive control

Value:

ca. 105.4 - ca. 113.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

In Vitro Irritancy score (IVIS) test substance

Value:

ca. 5.6 - ca. 10.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 5

Irritation parameter:

in vitro irritation score

Run / experiment:

In Vitro Irritancy score (IVIS) negative control

Value:

ca. 0 - ca. 2.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 6

Irritation parameter:

in vitro irritation score

Run / experiment:

In Vitro Irritancy score (IVIS) positive control

Value:

ca. 100.2 - ca. 100.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the observed results and applying the appropriate evaluation criteria it
was concluded, that N,N Dimethyldodecane-1-amide does not causes ocular corrosion
or severe irritation in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under
the test conditions chosen.

Executive summary:

The potential of N,N Dimethyldodecane-1-amide to cause serious damage to the eyes was assessed by a single topical application of 750 μL of the undiluted test substance to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test substance for 10 minutes followed by a 2-hours post-incubation period. In addition to the test substance a negative control (NC; de-ionized water) and a positive control (PC; 100% dimethylformamide) were applied to three corneas, each. Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance. In addition H&E-stained cross sections were evaluated for the irritation potential of the test substance.

The results were:

Test substance	Mean Opacity Value	Mean Permeability Value	MeanIn Vitro Irritancy Score
N,N Dimethyldodecane-1-amide	8.0	0.809	20.2
Negative control	1.8	0.00	1.8
Positive control	102.3	2.125	135.2

Based on the observed results and applying the appropriate  evaluation criteria it was concluded, that N,N Dimethyldodecane-1-amide does not causes ocular corrosion or severe irritation in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.