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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:
24 March 2014 - 10 April 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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
26 July 2013
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Reaction mass of 1,3,5-tris(5-isocyanatopentyl)-1,3,5-triazinane-2,4,6-trione and 1-{5-[3,5-bis(5-isocyanatopentyl)-2,4,6-trioxo-1,3,5-triazinan-1-yl]pentyl}-3,5-bis(5-isocyanatopentyl)-1,3,5-triazinane-2,4,6-trione
EC Number:
943-686-5
Molecular formula:
C21H30N6O6 + C35H50N10O10
IUPAC Name:
Reaction mass of 1,3,5-tris(5-isocyanatopentyl)-1,3,5-triazinane-2,4,6-trione and 1-{5-[3,5-bis(5-isocyanatopentyl)-2,4,6-trioxo-1,3,5-triazinan-1-yl]pentyl}-3,5-bis(5-isocyanatopentyl)-1,3,5-triazinane-2,4,6-trione
Test material form:
liquid: viscous
Details on test material:
- Storage conditions: sealed at room temperature (actual range 21.1-23.9°C)
- Physical appearance: colourless to clear pale yellow oily solution

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Hokkaido Livestock Corporation, Douo Plant, Hayakita Factory
- Characteristics of donor animals: donor animals were healthy cattle aged between 12 and 60 months
- Temperature during transport of ocular tissue: 3-13°C

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Test item: 750 µL
Positive control: 750 µL
Negative control: 750 µL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
3 replicates for the test item group, the positive control and the negative control each.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The eyeballs were macroscopically observed and examined for defects including corneal opacity, scratches and neovascularization. Corneas free of these abnormalities were used in the study. The corneas were dissected with a 2-3 mm rim of the sclera remaining and were immersed in Hank's balanced salt solution.
Corneas were mounted in corneal holders consisting of anterior and posterior chambers, which interfaced with the epithelial and endothelial sides of the cornea, respectively. Both chambers were filled with phenol red free MEM and the corneas were immersed in the medium and equilibrated in an incubator (MIR-262, Sanyo Electric Co., Ltd.) at 32°C +/- 1°C for 1 hour.
Following the equilibration, the baseline opacity value of each cornea was measured using the opacitometer and each cornea was macroscopically examined for defects. No corneas had an opacity value greater than 7 and there were no corneas with abnormalities.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 (with phenol red containing MEM)

- POST-EXPOSURE INCUBATION: yes, after completion of exposure and rinsing, the chambers were filled with phenol red free MEM and incubated at 32°C +/- 1°C for 2 hours.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: using an opacitometer (BASF-OP3.0, BASF)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV spectrophotometry (OD490)
- Others: macroscopic observations

SCORING SYSTEM: In Vitro Irritancy Score (IVIS), calculated from the opacity and permeability values: IVIS = opacity value/ (15*permeability OD490 value)

DECISION CRITERIA: see table 1 in 'any other information on materials and methods'

ACCEPTANCE CRITERIA:
- The IVIS of the positive control substance should be within 2 standard deviations of the latest historical control mean.
- The opacity and permeability values of the negative control substance should be less than the upper limits of historical control opacity and permeability values obtained from the bovine corneas treated with the negative control substance.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
Mean of 3 replicates
Value:
-1.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
Mean IVIS: 103.3
Other effects / acceptance of results:
- For the corneas treated with the test item, no individual corrected final opacity scores were calculated since the final opacity values were already <0. The mean corrected final opacity score was calculated to be -1.7. Permeability values were ranging from 0.000 to 0.002.

OTHER EFFECTS:
- No abnormalities (e.g. scratches, neovascularization, etc.) were macroscopically observed in any cornea during the measurements.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, results were within the historical range (mean opacity value: 2.2 and mean permeability value: 0.019)
- Acceptance criteria met for positive control: yes, results were within 2 standard deviation of the historical control mean (IVIS ranging from 42.6-130.6).

Any other information on results incl. tables

Table 2 Individual corneal opacity and permeability scores

 

 

Opacity

Permeability

Substance

 

Pre-exposure

Post-exposure

Final opacity

Corrected final opacity

OD490

OD490 change

Corrected OD490 change

 

Corneal No.

LUX

Opacity

LUX

Opacity

Negative control

1

970

2

964

2

0

-

0.011

0.003

-

 

4

953

2

947

3

1

-

0.011

0.003

-

 

6

957

2

951

3

1

-

0.007

-0.001

-

 

Mean

 

 

 

 

0.7

-

-

0.002

-

Test item

10

930

3

960

2

-1

-

0.010

0.002

0.000

 

11

943

3

964

2

-1

-

0.011

0.003

0.001

 

12

924

4

937

3

-1

-

0.012

0.004

0.002

 

Mean

 

 

 

 

-1.0

-1.7

-

-

0.001

Positive control

16

973

2

323

84

82

-

1.344

1.336

1.334

 

17

962

2

297

95

93

-

0.740

0.732

0.730

 

18

983

1

305

92

91

-

1.001

0.993

0.991

 

Mean

 

 

 

 

88.7

88.0

-

-

1.018

Calculation of scores:

Final opacity: (post exposure opacity) - (pre-exposure opacity)

Corrected final opacity: (mean final opacity) - (mean final opacity of negative control)

OD490 change: (OD490) - (blankOD490) [blank OD490 = 0.008]

Corrected OD490 change: (OD490 change - (mean OD490 change of negative control)

Opacity = ((baseline LUX)/(LUX) - 0.989)/0.025 [baseline LUX = 1000]

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In the Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline 437, D-370N induced a mean IVIS <3 (-1.7) after 10 minutes. Based on these results, the test item is not classified according to UN GHS.
Executive summary:

The eye hazard potential of D-370N was assessed in a Bovine Corneal Opacity and Permeability test (BCOP test), performed according to OECD guideline 437. Three corneas from healthy cattle were exposed to 750 µL of the test item (n=3), next to a negative control (n=3) and a positive control (dimethylformamide; n=3). Duration of treatment was 10 minutes with a post-incubation period of 2 hours. Results of the negative and positive controls were within the historical dataset. Since, it was shown that the negative control did not induce irritancy on the corneas, the test conditions were adequate and the test systems functioned properly. D-370N did not induce ocular irritation, resulting in a mean in vitro irritancy score of -1.7 after 10 minutes of treatment. Based on this result (IVIS < 3), the test item is not classified according to UN GHS.