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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:
weight of evidence
Study period:
2016-10-04 to 2016-12-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

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 (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-Benzyl-3-carbamoyl-pyridinium, chloride
Cas Number:
5096-13-9
Molecular formula:
C13H13ClN2O
IUPAC Name:
1-Benzyl-3-carbamoyl-pyridinium, chloride
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: obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany
- Number of animals: no data
- Characteristics of donor animals (e.g. age, sex, weight): no data
- Storage, temperature and transport conditions of ocular tissue: On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories.
- Time interval prior to initiating testing: Immediately after arrival of the eyes, cornea preparation was initiated.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.
- Indication of any antibiotics used: no data

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µl of test item preparation
- Concentration (if solution): 20 % concentration in 0.9 % physiological saline NaCl

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): 0.9 % physiological saline NaCl
- Lot/batch no. (if required): B. Braun Melsungen, lot no. 1406804, expiry date: 05/2017
Duration of treatment / exposure:
4 hours ± 5 minutes incubation at 32 +/- 1 °C
Duration of post- treatment incubation (in vitro):
90 minutes at 32 +/- 1 °C
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS + QUALITY CHECK OF THE ISOLATED 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 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 +/- 1 °C.

NUMBER OF REPLICATES: 3 per test group (test item, positive and negative control)

NEGATIVE CONTROL USED: yes, physilogical saline 0.9 % NaCl

SOLVENT CONTROL USED (if applicable): see negative control

POSITIVE CONTROL USED: yes, imidazole 20% in physiological saline 0.9% NaCl; imidazole from Sigma, lot no. SLBK9670V, expiry date: 01/2017

TREATMENT METHOD: open chamber
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. 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. The medium was removed from the anterior chamber and replaced with the test item or control.

APPLICATION DOSE AND EXPOSURE TIME
750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). Corneas were exposed for 4 hours ± 5 minutes incubation at 32 +/- 1 °C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed. Also, each cornea was observed visually and pertinent observations were recorded.

POST-EXPOSURE INCUBATION:
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 +/- 1 °C. Then the medium from the posterior chamber was removed and its optical density was determined.

METHODS FOR MEASURED ENDPOINTS:
- The optical density was determined at 490 nm (OD490) using a spectrophotometer (Jenway 6405 UV/VIS).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
- IVIS ≤ 3: UN GHS No Category
- IVIS > 3 to ≤ 55: No prediction can be made
- IVIS > 55: UN GHS Category 1

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean score of 3 replicates
Value:
69.38
Vehicle controls validity:
valid
Negative controls validity:
valid
Remarks:
The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
Positive controls validity:
valid
Remarks:
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

Any other information on results incl. tables

Evaluation of Results

The following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer:

 

Opacity = (I0/I - b) / a

with a = 0.025 and b = 0.9894

 

The value I0 is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated periodically.

The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.

The mean OD490 for the blank cuvettes was calculated. The mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:

Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490

The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

The following formula was used to determine the in vitro irritation score (IVIS):

IVIS = mean opacity value + (15 x mean permeability OD490 value)

The IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given in Table 1:

Table 1:  Evaluation of the BCOP Assay

IVIS

UN GHS

≤ 3

No Category

> 3; ≤ 55

No prediction can be made

> 55

Category 1

An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made.For this purpose further testing with another suitable method is required.

Individual Results Data

Table 2: Opacity

Cornea No.

Test item

Initial Opacity

Final Opacity

Change of Opacity Value

Corrected Opacity Value

1

Negative Control

0.84

1.60

0.76

 

2

0.74

2.01

1.27

 

3

0.81

1.64

0.83

 

MV

0.80

1.75

0.95

 

4

Positive Control

1.42

99.72

98.30

97.35

5

2.05

116.82

114.77

113.82

6

2.05

118.43

116.39

115.43

MV

1.84

111.66

109.82

108.87

7

Test item

-0.06

69.57

69.63

68.68

8

0.42

52.87

52.45

51.50

9

1.38

78.87

77.49

76.53

MV

0.58

67.11

66.52

65.57

MV = Mean value

 

Table 3: Permeability

Cornea No.

Test item

OD490

Corrected OD490 Value

1

Negative Control

0.015

 

2

0.026

 

3

0.014

 

MV

0.018

 

4

Positive Control

1.975

1.957

5

2.175

2.157

6

2.245

2.227

MV

2.132

2.113

7

Test item

0.191

0.173

8

0.358

0.340

9

0.267

0.249

MV

0.272

0.254

MV = Mean value

 

Table 4: In Vitro Irritation Score

Cornea No.

Test item

Corrected Opacity

Corrected OD490 Value

IVIS

1

Negative Control

0.76

0.015

 

2

1.27

0.026

 

3

0.83

0.014

 

MV

0.95

0.018

1.23

4

Positive Control

97.35

1.957

 

5

113.82

2.157

 

6

115.43

2.227

 

MV

108.87

2.113

140.57

7

Test item

68.68

0.173

 

8

51.50

0.340

 

9

76.53

0.249

 

MV

65.57

0.254

69.38

MV = Mean value

 

Table 5: Historical mean In Vitro Irritation Score of the Positive Control

 

IVIS

Positive Control

Mean Value (MV)

126.72

Standard Deviation (SD)

17.00

MV – 2xSD

92.72

MV + 2xSD

160.72

Number of Replicates providing Historical Mean: 17

 

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Based on the mean in vitro irritation score of 69.38 obtained in the bovine corneal opacity and permeability assay (BCOP, OECD 437), the test substance needs to be classified as Eye Dam. 1 (H318) in accordance with Regulation (EC) No 1272/2008 (CLP).
Executive summary:

The eye irritancy potential of 1-Benzyl-3-carbamoyl-pyridinium, chloride was investigated in the bovine corneal opacity and permeability assay (OECD 437). The test item was suspended with physiological saline 0.9% NaCl to give a 20% concentration. All three corneas treated with 1-Benzyl-3-carbamoyl-pyridinium, chloride showed strong opacity of the tissue. The mean in vitro irritation score in this test was 69.38. The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control. Based on the results obtained the test substance needs to be classified as Eye Dam. 1 (H318) in accordance with Regulation (EC) No 1272/2008 (CLP).