Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Sep 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
26 July 2013
Deviations:
yes
Remarks:
no data on values (ranges) of historical positive and negative control are provided in the report
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Test material

Constituent 1
Reference substance name:
Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates
EC Number:
306-227-4
EC Name:
Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates
Cas Number:
96690-34-5
Molecular formula:
C4H11O4P to C8H20O7P2 as representative molecular formula of the composition as specified in section 1.2
IUPAC Name:
Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Attenberger Fleisch GmbH & Co. KG, Germany
- Storage, temperature and transport conditions of ocular tissue: On the test day, fresh eyes were collected from the slautherhouse 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: Yes

Test system

Vehicle:
other: sesame oil
Controls:
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied in the test: 750 µL
- Concentration: 20% in sesame oil

VEHICLE
- Substance: sesame oil (Caelo)
- Amount(s) applied in the test: 750 µL
- Lot/batch no.: 12054501
Duration of treatment / exposure:
4 h ± 5 min
Number of animals or in vitro replicates:
number of eyes for the test item: 3
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.

QUALITY CHECK OF THE ISOLATED CORNEAS
Before the corneas were mounted in corneal holders with the endothelial side against the O-ring of the posterior chamber, a visual check was examined for defects and any defective cornea was discarded.

NUMBER OF REPLICATES
3 eyes for each test group

NEGATIVE CONTROL USED
0.9% physiological saline (AlleMan Pharma, Lot/batch no.: 111214)

SOLVENT CONTROL USED
sesame oil (Caelo, Lot/batch no.: 12054501)

POSITIVE CONTROL USED
20% Imidazole (Sigma, Lot/batch no.: 109K5306V) solution in physiological saline (0.9% NaCl)

APPLICATION DOSE AND EXPOSURE TIME
750 µL and 4 h ± 5 min for each test group

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times with Minimal Essential Medium (MEM)
- POST-EXPOSURE INCUBATION: 90 min

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: decreased light transmission with the aid of a microtiter plate reader (OD490)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
According to OECD 437, liquids are usually tested undiluted and corneas are exposed to the liquids for 10 min. However, the test item was too viscous to be directly applied to the corneas. On the demand of the sponsor, the test item was suspended in sesame oil and tested according to the protocol for solids. Therefore, imidazole was used as positive control, sesame oil was used as an additional negative control and the exposure period was expanded to 4 h instead of 10 min.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
mean value of 3 eyes
Run / experiment:
4 h of exposure
Value:
222.1
Positive controls validity:
valid
Other effects / acceptance of results:
Since the IVIS was 222.1, the test substance was defined as a corrosive. The IVIS of the positive control was 158.55, thus higher than that of the historical control values (out of the two standard deviations of the current historical mean), but still regarded valid.
No further lesions of the isolated eyes were reported.

Any other information on results incl. tables

DATA EVALUATION

- Calculation of opacity values:

The opacity value was calculated according to the following steps:

i) opacity change = final opacity – initial opacity

ii) corrected opacity change = opacity change – mean opacity change of the negative control

iii) mean opacity value = mean of all corrected opacity changes per group

 

- Calculation of OD490 (optical density at 490 nm) values:

The permeability value was calculated according to the following steps:

iv) OD490 change = OD490 – mean blank 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.

v) corrected OD490 change = OD490 change – mean OD490 change of negative control

vi) mean OD490 value = mean of all corrected OD490 changes per group

 

- Calculation of the IVIS (in-vitro irritancy score) values:

The In-Vitro Irritancy Score (IVIS) was calculated as follows:

IVIS per cornea per group = mean corrected opacity change + (15 x mean corrected OD490 change)

 

- Evaluation criteria:

Test substance with an IVIS ≥ 55.1 was regarded as severe irritant/corrosive and labelled Category 1.

 

- Acceptance criteria:

OECD 437: “A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean, which is to be updated at least every three months, or each time an acceptable test is conducted in laboratories where tests are conducted infrequently (i.e., less than once a month). The negative or solvent/vehicle control responses should result in opacity and permeability value that are less than the establisher upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control.”

RESULTS

Table 1. Opacity scores

Treatment group

Cornea no.

Initial opacity

Final opacity

Opacity change

Corrected opacity change

Mean corrected opacity change

Test substance (20% in sesame oil)

7

8

9

3

4

4

195

216

210

192

212

206

188.33

208.33

202.33

199.67

Negative control (0.9% NaCl)

1

2

3

4

4

4

7

9

9

3

5

5

NA

NA

NA

NA

Negative control (sesame oil)

10

11

12

4

5

5

8

10

7

4

5

2

NA

NA

NA

NA

Positive control (20% imidazole in 0.9% NaCl)

4

5

6

5

5

5

151

174

136

146

169

131

141.67

164.67

126.67

144.33

NA = not applicable

 

Table 2. Permeability score

Treatment group

Cornea no.

OD490 value

Corrected OD490 value

Mean corrected OD490 value

Test substance (20% in sesame oil)

7

8

9

1.355

1.618

1.632

1.316

1.579

1.593

1.496

Negative control (0.9% NaCl)

1

2

3

0.074

0.098

0.107

NA

NA

NA

NA

Negative control (sesame oil)

10

11

12

0.043

0.020

0.053

NA

NA

NA

NA

Positive control (20% imidazole in 0.9% NaCl)

4

5

6

1.034

0.878

1.211

0.941

0.785

1.118

0.948

NA = not applicable

 

Table 3. In vitro irritancy score (IVIS)

Treatment group

Cornea no.

Corrected opacity change

Corrected OD490

IVIS

per group

mean

Test substance (20% in sesame oil)

7

8

9

188.33

208.33

202.33

1.316

1.579

1.593

222.11

Positive control (20% imidazole in 0.9% NaCl)

4

5

6

141.67

164.67

126.67

0.941

0.785

1.118

158.55

 

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
CLP: Cat. 1, H318