Alfalfa, ext.

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 April 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

Test guideline 1

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:

Guideline version from 9 October 2017

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Alfalfa, ext.

EC Number:

281-984-0

EC Name:

Alfalfa, ext.

Cas Number:

84082-36-0

IUPAC Name:

Alfalfa, ext.

Test material form:

liquid: viscous

Remarks:

Alfalfa, ext was observed to be a very thick dark coloured paste at room temperature.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AL01
- Expiration date of the lot/batch: 10.09.2019
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: approximately at 4 °C in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: water solubility 5.35 mg/L at 25C
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not available
- Preliminary purification step (if any): not available
- Final dilution of a dissolved solid, stock liquid or gel: not available
- Final preparation of a solid: not available

OTHER SPECIFICS: none

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): adult cattle, 12-60 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): transported to the test facility over ice packs on the same day of slaughter
- Time interval prior to initiating testing: corneas were prepared immediately on arrival from abattoir
- Indication of any existing defects or lesions in ocular tissue samples: only damage free corneas were used
- Indication of any antibiotics used: 100 IU/mL penicillin and 100 IU/mL streptomycin

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): not applicable; applied undiluted

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Triplicate

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing Hanks’ Balanced Salt Solution (HBSS) until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects.

QUALITY CHECK OF THE ISOLATED CORNEAS
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer.

NUMBER OF REPLICATES
Three corneas were randomly allocated to the test item, negative and positive control.

NEGATIVE CONTROL USED
Sodium chloride, 0.9 % w/v.

SOLVENT CONTROL USED (if applicable)
Not applicable

POSITIVE CONTROL USED
Ethanol, >99.8%

APPLICATION DOSE AND EXPOSURE TIME
0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1ºC for 10 minutes.

TREATMENT METHOD: closed chamber.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

POST-EXPOSURE INCUBATION: 32 ± 1°C for 120 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader] (OD492)
- Others (e.g, pertinent visual observations, histopathology): (please specify):
The condition of the cornea was visually assessed post treatment and post incubation. No histopathology was performed.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

DECISION CRITERIA:
For an acceptable test the following positive control criterion should be achieved:
Neat ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during 2016 for this testing facility. Therefore, the In Vitro Irritancy Score should fall within the range of 31.6 to 58.7.

For an acceptable test the following negative control criteria should be achieved:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2016 for bovine corneas treated with the respective negative control. When testing liquids the negative control limit for opacity should be =3.0 and for permeability =0.077.

The test item was classified according to the following prediction model:
IVIS Classification
= 3: No category. Not requiring classification to UN GHS or EU CLP;
> 3: =55 No prediction of eye irritation can be made;
> 55: Category 1. UN GHS or EU CLP Causes serious eye damage.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

5.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Any other information on results incl. tables

The In Vitro irritancy scores:

Treatment	In Vitro Irritancy Score
Test Item	5.9
Negative Control	0.0
Positive Control	45.4

Applicant's summary and conclusion

Interpretation of results:

other: No prediction can be made

Conclusions:

Based on the results from the Bovine Corneal Opacity and Permeability (BCOP) test, conducted according to OECD test guideline 437 and to GLP on Alfalfa, ext. no prediction of eye irritation can be made.

Executive summary:

The Bovine Corneal Opacity and Permeability (BCOP) test, conducted according to OECD test guideline 437 and to GLP, was performed to evaluate the eye hazard potential of Alfalfa, ext.

The undiluted test item was applied for 10 minutes then rinsed followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

The In Vitro Irritancy Score for Alfalfa, ext. was determined at 5.9; therefore, no prediction of eye irritation can be made.