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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:
2017-05-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (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)
Version / remarks:
2013-07-26
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2010-12-08
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N'-[(methylimino)bis(trimethylene)]bis(stearamide)
EC Number:
236-793-7
EC Name:
N,N'-[(methylimino)bis(trimethylene)]bis(stearamide)
Cas Number:
13483-58-4
Molecular formula:
C43H87N3O2
IUPAC Name:
N-{3-[methyl(3-octadecanamidopropyl)amino]propyl}octadecanamide

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
Source OF fresh bovine corneae are animals is cattle (species Bos primigenius Taurus).
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported within 1 hour to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a cooled container. Then, the corneae were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 h.

PREPARATION
Clean and sterile cornea holders were kept in the in-cubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bi-carbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C. The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.

After the arrival of the corneae, they were examined and only corneas which were free from damages were used. The corneae were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneae showed tissue damage; therefore, all corneae were used. For each treatment group (negative control solution, test item and positive con-trol solution), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 µL negative control solution, positive control solution and a defined amount of test item were applied to each replicate.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test item was applied onto the epithelium so that the cornea was completely and homogeneously covered.
Replicate Amount of neat pestled test item
1 535.8 mg
2 563.3 mg
3 560.6 mg
Duration of treatment / exposure:
Exposure time of the test item and the controls on the corneas was 4 hours at 32 ± 1 °C.
Observation period (in vivo):
Exposure time of the test item and the controls on the corneas was 4 hours at 32 ± 1°C.
Number of animals or in vitro replicates:
3 corneae per group (test item, negative control, positive control)
Details on study design:
REMOVAL OF TEST ITEM
After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded.
The cMEM without phenol red was then removed from the front chamber, and 1 mL so-dium fluorescein solution (concentration: 5 mg/mL) was added to the front chamber.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the perme-ability of the cornea was measured as optical density of the liquid with a spectrophotome-ter at 492 nm.

SCORING SYSTEM
The IVIS of each replicate of the negative control was calculated from the following equation:
IVIS = opacity difference (opacity of treated corneae - opacity of negative control) + (15 x corrected OD492 value)

SPECIFIC EQUIPMENT
Opacitometer BASF OP 3.0 (BASF)
Cornea holders (Duratec Analysentechnik GmbH)

VALIDITY
According to the OECD guideline 437 (26 July 2013), the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean.
The negative control has to show an IVIS ≤ 3.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
0.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
VALIDITY
Parameter Criterion Found Assessment
IVIS of negative control ≤ 3 0.55 OK
HBSS-solution
IVIS of positive control 2.75 - 166.63 108.80 OK
20% imidazole solution

Values for negative and positive controls were within the range of historical data of the test facility.
Therefore, the test system was acceptable and valid.

Any other information on results incl. tables

Results afer 4 h incubation

Test group

 

Opacity difference corrected

 

Permeability at 492 nm*

 

In vitro

Irritation

Score

Mean

in vitro

Irritation

Score

Relative SD

of IVIS

Negative control

-

-

0.58

0.55

25.02 %

-

-

0.40

-

-

0.68

Positive

control

91.71

2.3362

126.75

 108.80

 16.75 %

68.35

1.4646

90.32

75.20

2.2746

109.32

Test

substance

0.15

0.0021

0.12

 0.70

76.33 %

0.39

0.0286

0.82

0.23

0.0626

1.17

- The mean opacity difference of the negative control was 0.35.

- For the blank, the mean optical density at 492 nm was 0.038.

- The values for Permeability at 492 nm were all corrected by subtracting the mean blank value.

- For the negative control, the mean Permeability at 492 nm was 0.0138.

- For the positive control, the all values for the Permeability at 492 nm were obtained by measurement of a fivefold diluted solution and multiplication of the absorbances with factor 5.

- The high relative standard deviation of the IVIS of test item is due to mathematical reasons, as the respective means are very small.

COMPARISON WITH HISTORICAL DATA

In the following table, the means of the negative control and positive control of all experiments which were performed at the test facility up to 11. May 2017 are stated and compared with the values which were found in this study.

 

Negative Control

Positive Control

Mean IVIS

1.99

119.69

Standard Deviation IVIS

1.03

23.47

Range of IVIS (validity)

≤ 3

72.75 – 166.63

Study 17031603G850

0.55

108.80

Mean Opacity

1.73

82.38

Standard Deviation

Opacity

1.01

18.45

Range of Opacity

-1.86 – 4.08

42.92 – 133.11

Study 17031603G850

0.35

78.42

Mean Permeability

0.02

2.52

Standard Deviation Permeability

0.02

1.02

Range of Permeability

-0.01 – 0.10

0.75 – 5.89

Study 17031603G850

0.01

2.03

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In the Bovine Corneal Opacity and Permeability Test, the test item N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) has an IVIS of 0.7.
This result lies within the range of IVIS ≤ 3 and, according to OECD Guideline 437 (26 July 2013), this substance requires no classification for eye irritation or serious eye damage.
Executive summary:

An in-vitro eye irritation study in accordance with OECD Guideline 437, with N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) was performed using the Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular corrosives and Severe Irritants.

Bovine corneas, collected from slaughtered cattle which were between 12 and 60 months old, were used for the test.

The test item N,N'-[(methylimino)bis(trimethylene)]bis(stearamide) was tested pure. It was pestled and brought onto the cornea of a bovine eye, which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for4 hours at 32 ± 1 °C. After removal of the test item opacity and permeability values were measured.

HBSS-solution was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) is 0.55.

20% imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and falls within two standard deviations of the current historical mean.

The calculated IVIS (in vitro irritancy score) is 108.80.

 

Under the conditions of this study, the test item N,N'-[(methylimino)bis(trimethylene)]bis(stearamide) showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 0.70.

Since the IVIS of N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) has a value of ≤ 3, according to OECD Guideline 437 (26 July 2013), this substance requires no classification for eye irritation or serious eye damage.

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