Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-03-06 to 2017-05-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
July 26, 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
December 09, 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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: Hubert Bahlmann GmbH & Co. Versandschlachterei Spezialmischfutterwerk KG, 49699 Lindern, Germany
- Number of animals: at least five, three corneae for each concentration (negative control, positive control, treatment)
- Characteristics of donor animals (e.g. age, sex, weight): between 6 and 12 month old cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution 2 (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL.
- indication of any existing defects or lesions in ocular tissue samples: Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 µg/mL

Test system

Vehicle:
unchanged (no vehicle)
Remarks:
20% suspension in 0.9% sodium chloride solution (w/v)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% suspension in 0.9% sodium chloride solution (w/v)

Duration of treatment / exposure:
240 min
Duration of post- treatment incubation (in vitro):
90 ± 5 min
Number of animals or in vitro replicates:
three corneae per treatment
Details on study design:
SELECTION AND PREPARATION OF CORNEAE
Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse. To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution 2 (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL 3 . Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used.

NUMBER OF REPLICATES
Three coneae per concentration

SOLVENT CONTROL USED (if applicable)
0.9 % sodium chloride solution (w/v)

POSITIVE CONTROL USED
20% Imidazole in 0.9% sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME
750 µL of each solution was applied for 240 min

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The IVIS cut-off values for identifying test chemicals as inducing serious damage (UN GHS Category 1) and test chemicals not requiring classification for irritation or serious eye damage (UN GHS No Category):
≤ 3: No Category
> 3 and ≤ 55: No prediction can be made
> 55: Category 1

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
2.097
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
1
Value:
-1.99
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Run / experiment:
1
Value:
-0.007
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The negative or solvent/vehicle control responses should result in opacity and permeability values, that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control. (see Table 1)
- Acceptance criteria met for positive control: Yes. The positive control should give an IVIS that falls within two standard deviations of the current historical mean. (see Table 1)
- Range of historical values if different from the ones specified in the test guideline: No

Any other information on results incl. tables

Table 1: Historical control data

 

Parameter

Mean

Standard deviation

Lower limit of acceptance

(mean-2*SD)

Upper limit of acceptance

(mean+2*SD)

NaCl 0.9 %

IVIS Opacity

0.812 0.513

0.762 0.788

-0.712 -1.064

2.336 2.089

 

Permeability

0.020

0.012

-0.005

0.045

Imidazol

20%

IVIS Opacity

86.039

63.051

10.607

10.709

64.826

41.634

107.253

84.468

 

Permeability

1.533

0.463

0.607

2.458

Table 2: Summary of results after 240 min incubation

Test group

Opacity

Permeability at 490 nm

In vitro Irritation score

Mean In vitro Irritation Score

In vitro Irritation Scale

Negative control

0.917

0.010

1.067

 

Not severely irritating

-0.797

0.011

-0.632

 

-0.358

0.010

-0.208

0.076

Positive control

73.505

1.403

94.629

 

Severely irritating

49.323

1.204

67.462

 

54.463

1.372

75.122

79.071

Test item

-2.032

0.006

-2.043

 

Not severely irritating

-2.031

0.005

-2.027

 

-2.151

-0.010

-2.222

-2.097

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In the present study with alpha methyl glucoside conducted according to OECD guideline 437, three corneae for each treatment, i.e. negative control, positive control (20% imidazol in 0.9 % sodium chloride solution (w/v)) and treatement (20% alpha methyl glucoside in 0.9 % sodium chloride solution (w/v)) were exposed for 240 min. Opacity and permeability revealed values < 0 and the IVIS was < 3 (2.097), therefore, the test item is not classified according to UN GHS classification.
Executive summary:

In the present study with alpha methyl glucoside conducted according to OECD guideline 437, three corneae for each concentration, i.e. negative control, positive control (20% imidazol in 0.9 % sodium chloride solution (w/v)) and treatement (20% alpha methyl glucoside in 0.9 % sodium chloride solution (w/v)) were exposed for 240 min. Subsequently the opacity was determined using a opacitometer and the corneal permeability was detected with the fluorescein method.

The positive control increased opacity and permeability (mean in vitro score 79.07) corresponding to a classification as irreversible effects to the eye.

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro score 0.00).

Since, the results of the test item for opacity and permeability revealed values < 0 and the IVIS was < 3 (2.097) the test item is not classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).