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Diss Factsheets

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:
15 Mar 2018
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 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:
adopted in 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Test material

Constituent 1
Reference substance name:
Reaction product of C16-18 (even numbered) alcohols with reaction products of 1,3,5-Triazine, 2,4,6,-triamine, polymer with formaldehyde, methylated
EC Number:
947-918-6
Molecular formula:
not applicable, UVCB substance.
IUPAC Name:
Reaction product of C16-18 (even numbered) alcohols with reaction products of 1,3,5-Triazine, 2,4,6,-triamine, polymer with formaldehyde, methylated
Test material form:
solid

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported to the laboratory in Hanks´ Balanced Salt Solution (HBSS) containing Pen/strep on ice.
- Time interval prior to initiating testing: The corneae preparation started on the same day after delivery of the eyes and were directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.

Test system

Vehicle:
other:
Remarks:
test substance was moistened with physiological saline 0.9% NaCl
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL

NEGATIVE CONTROL
- Amount applied: 750 µL
- Concentration: 0.9% NaCl

POSITIVE CONTROL
- Amount applied: 750 µL
- Concentration: 20% in physiological saline (0.9% NaCl)
Duration of treatment / exposure:
4 h ± 5 min at 32 ± 2.6 °C
Number of animals or in vitro replicates:
triplicates for each treatment and control group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with Roswell Park Memorial Institute medium (RPMI, without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 2.6 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the equilibration period, an initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay.


TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: corneae were washed at least three times with MEM (containing phenol red)


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (BASF-OP3.0, Duratec GmbH)
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a spectrophotometer (Jenway 6405 UV/VIS) at 490 nm (OD490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS), IVIS = mean opacity value + (15 x mean OD490 value)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as serious eye damage and labelled Category 1 according to CLP/EHS/GHS.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
4 h
Value:
0.18
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:
With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.74). The positive control (imidazole 20%) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 149.42).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control resulted in opacity and permeability values that were less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- Acceptance criteria met for positive control: The positive control resulted in an IVIS which was within two standard deviations of the current historical mean.

Any other information on results incl. tables

Table 3. In Vitro Irritancy Scores

Cornea No.

Test Item

Corrected Opacity

Corrected OD490 Value

IVIS

1

 

0.71

0.024

 

2

Negative

0.37

0.015

 

3

Control

0.37

0.012

 

MV

 

0.49

0.017

0.74

4

 

88.86

2.838

 

5

Positive

116.87

2.198

 

6

Control

129.66

2.488

 

MV

 

111.80

2.508

149.42

7

 

-0.26

-0.004

 

8

Test Item

0.88

0.019

 

9

 

-0.27

-0.003

 

MV

 

0.12

0.004

0.18

MV = mean value

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
Under conditions of the Bovine Corneal Opacity and Permeability Test (BCOP) the test subtance was not irritating to the eye. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 0.18