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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:
11 December 2017 - 12 December 2017
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:
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:
9 October 2017
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Appearance: Off-white powder
Test item storage: At room temperature
Specific details on test material used for the study:
pH: 6.07-6.62

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: young cattle, obtained from the slaughterhouse Vitelco, 's-Hertogenbosch, The Netherlands.
- Storage, temperature and transport conditions of ocular tissue: eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Subsequently, eyes were collected an transported in physiological saline in a suitable container under cooled conditions.
- The eyes were checked for unacceptable defects and those exhibiting defects were discarded.
- Isolated corneas were stored in a petri dish with cMEM containing 1% (v/v) L-glutamine and 1% (v/v) Fetal Bovine Serum.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 320.0 to 339.8 mg

POSITIVE CONTROL
- Amount applied: 750 µL

NEGATIVE CONTROL
- Amount applied: 750 µL
Duration of treatment / exposure:
240 ± 10 minutes
Duration of post- treatment incubation (in vitro):
90 ± 5 minutes in sodium-fluorescein for permeability measurements
Number of animals or in vitro replicates:
3 for the negative control, the positive control and the treatment group each.
Details on study design:
TREATMENT METHOD: The medium from the anterior compartment was removed and 750 µL of the negative control and the positive control were introduced onto the epithelium of the cornea. The test substance was weighed in a glass tube and applied directly on the corneas in such a way that the cornea was completely covered (320.0 to 339.8 mg). The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies).

- POST-EXPOSURE INCUBATION: 90 ± 5 minutes at 32±1 °C in sodium-fluorescein for permeability determinations.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity meter (OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (TECAN Infinite® M200 Pro Plate Reader, OD490). OD490 values of less than 1.500 were used in the permeability calculation.
- Other: possible pH effects of the test substance on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA (see table 1):
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces IVIS >55 is defined as a corrosive or severe irritant (UN GHS: category 1);
For a test substance that induces an IVIS >3 and ≤55, no prediction on irritant potency can be made.

ACCEPTABILITY CRITERIA:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean of 3 replicates
Value:
< 0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
IVIS: -2.7-2.6
Positive controls validity:
valid
Remarks:
IVIS: 110-139
Other effects / acceptance of results:
- The corneas treated with the test substance showed opacity values between -1.5 and -1.0.
- Permeability values were ranging from 0.003 to 0.036
- IVIS scores were -0.9, -0.9 and -0.8 (n=3), resulting in a mean IVIS of -0.9. Since the IVIS scores were < 3, the substance is considered to be non irritant.
- The corneas were clear after 240 minutes of treatment and no pH effect of the test item was observed on the rinsing medium.

ACCEPTANCE OF RESULTS (see table 2 for historical data):
- Acceptance criteria met for negative control: yes, responses for opacity and permeability were less than the upper limits of the historical data range (IVIS ranging from -2.7 to 2.6).
- Acceptance criteria met for positive control: yes, results were within historical range (IVIS ranging from 110 to 139). Corneas were turbid after 240 minutes of treatment.

Any other information on results incl. tables

Table 2 Historical Control Data for the BCOP Studies

 

Negative control

Positive control

 

Opacity

Permeability

In vitro Irritancy Score

In vitro Irritancy Score

Range

-5.4 – 5.2

-0.010 – 0.205

-5.3 – 5.3

86.5 - 211.4

Mean

0.50

0.02

0.72

135.88

SD

1.83

0.02

1.89

26.52

n

136

136

136

137

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of Nov 2014 to Nov 2017.

Treatment

Final Opacity*

Final OD490*

In vitroIrritancy Score#

 

Negative control

0.1

0.010

0.8

2.5

0.009

0.5

-2.5

-0.011

-0.3

 

Positive control

122

1.391

139

137

1.807

122

126

1.781

110

 

Test item

-1.5

0.036

-0.9

-1.0

0.003

-0.9

-1.3

0.034

-0.8

#  In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

*  Positive control and test item are corrected for the negative control.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) 1272/2008
Conclusions:
Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline 437 and GLP principles, D6-Noreth is considered to be non-irritant for the eyes. The test item is not classified for eye irritation or serious eye damage according to GHS and according to Regulation (EC) 1272/2008.