Registration Dossier

Diss Factsheets

Toxicological information

Eye irritation

Currently viewing:

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11.06.2018 - 09.08.2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study was performed in compliance with the Principle of Good Laboratory Practice, confirmed by Statement of GLP Compliance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

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)
Principles of method if other than guideline:
None known.
GLP compliance:
yes (incl. QA statement)

Test material

1
Reference substance name:
Rhatany, Krameria triandra, ext.
EC Number:
283-919-1
EC Name:
Rhatany, Krameria triandra, ext.
Cas Number:
84775-95-1
Molecular formula:
not available
IUPAC Name:
Rhatany, Krameria triandra, ext.
Test material form:
liquid: viscous
Specific details on test material used for the study:
Name Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction
Batch no. PES180014
Appearance reddish brown viscous substance
Composition Krameria triandra extractobtained from Rhatany root by hydroalcoholic extraction
Purity not applicable, UVCB
Homogeneity inhomogeneous, warm up to about 60°C and stir
Expiry date Jan. 2019
Storage Fridge (2 - 8 °C)
CAS No. 84775-95-1
EINECS-No. 283-919-1
Chemical Class not stated
Volatility unknown
pH-value unknown
Stability H2O: 96h; EtOH: 96h; acetone: 96h; CH3CN: 96h; DMSO: 96h
Solubility H2O: unknown; EtOH: >1 g/L; acetone: >1 g/L; CH3CN: >1 g/L; DMSO: >1 g/L
Surface activity no

Test animals / tissue source

Species:
other: Bos primigenius Taurus (fresh bovine corneas)
Details on test animals or tissues and environmental conditions:
Origin:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour (Exp. 1 and Exp. 1c) or 1 hour and 30 minutes (Exp. 1b).

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Details on study design:
Preparation:
The test item is a viscous liquid substance. It was tested directly, without dilution or preparation of a solution. On the day of the test, the test item was warmed up to about 60°C and stirred to achieve homogeneity and that it became pipettable.
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C.
The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.
After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

Method Description:
After the initial incubation, the medium was completely changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item and positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 µL negative control solution, test item and positive control were applied to each replicate to the epithelial side of the cornea.
According to the characteristics of the test item, the following treatment procedure was performed:

Open Chamber Method:
The “open chamber-method” is used for solid or viscous substances.
In order to apply the test item, the window-locking ring and glass window of the anterior chamber was unscrewed to remove the glass disc. The test item was drawn into a pipette tip, cooled down and before it hardened the test item was applied directly onto the cornea. Exposure time of controls and test item on the corneas was 10 minutes at 32 ± 1 °C.

After thorough rinsing the anterior chamber with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red and the corneaholder were stored for additional 2 hours at 32 ± 1 °C (post-incubation). Then, the final illuminance value of each cornea was recorded.

Permeability Test
After the recording of the final opacity value, the cMEM without phenol red was removed from both chambers. The posterior chamber, which interfaces with the endothelial side of the cornea was filled with fresh cMEM. Then 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas.
A sodium fluorescein solution with a concentration of 4 mg/mL was used.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C in a horizontal position. After incubation, the content of the posterior chamber was thoroughly mixed and pipetted in a 96-well plate. Then, its optical density at 492 nm is measured with the microtiter plate photometer.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
Experiment 1
Value:
2.86
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
Experiment 1b
Value:
4.49
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
Experiment 1c
Value:
3.02
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Assessment:
Classification Scheme
IVIS UN GHS
≤ 3 No category
> 3 and ≤ 55 No prediction can be made
> 55 Eye damage Category I

In all experiments, no signs of eye irritation could be observed in the negative control.
The positive control induced serious eye damage, which would be classified as GHS category I.
In the first experiment under the conditions of the study, the test item Krameria triandra extract showed no effects on the cornea of the bovine eye. The calculated mean IVIS was 2.86 (Exp. 1).
In the second and third experiment under the conditions of this study, the test item Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction showed effects on the cornea of the bovine eye. The calculated mean IVIS was 4.49 (Exp. 1b) and 3.02 (Exp. 1c).
The two out of three approach lead to the following assessment:
According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage with the BCOP study only. In this case no prediction can be made.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
As the test item itself is inhomogeneous, the result of the study has to be considered equivocal.
Conclusions:
This in vitro study was performed to assess corneal damage potential of Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction by quantitative measurements of changes in opacity and permeability in a bovine cornea.
The test item Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction was brought onto the cornea of a bovine eye which previously had been incu-bated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.
The test item was tested neat.
In the first experiment under the conditions of the study, the test item Krameria triandra extract showed no effects on the cornea of the bovine eye. The calculated mean IVIS was 2.86 (Exp. 1).
In the second and third experiment under the conditions of this study, the test item Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction showed effects on the cornea of the bovine eye. The calculated mean IVIS was 4.49 (Exp. 1b) and 3.02 (Exp. 1c).
The two out of three approach lead to the following assessment:
According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage with the BCOP study only. In this case no prediction can be made.
The negative control (HBSS) and the positive control (undiluted dimethylformamide) have met the validity criteria.
As the test item itself is inhomogeneous, the result of the study has to be considered equivocal.
Executive summary:

This in vitro study was performed to assess the corneal damage potential of Krameria triandra extract obtained from Rhatany root by hydroalcoholic extractionby quantitative measurements of changes in opacity and permeability in a bovine cornea. The study was performed for regulatory purposes.

The BCOP test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. Both measurements are used to calculate an “In Vitro Irritancy Score (IVIS)”, which is used to classify the test item in the UN Globally Harmonised System (GHS).

The BCOP test method uses isolated corneas from the eyes of freshly slaughtered cattle. Corneal opacity is measured quantitatively as the amount of light transmission through the cornea. Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber. Test item is applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder.

Three valid experiments were performed.

The result of the first experiment (Exp. 1) was equivocal, because two of the three replicates gave discordant predictions from the mean value. Therefore a second experiment was performed. The result of the second run (Exp. 1b) was unequivocal, but gave a non-concordant prediction from the first testing run. This is why a final testing (Exp. 1c) run was conducted to resolve equivocal predictions. The third test run confirmed the result of the second test run.

 

In all experiments bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old.

The test item Krameria triandra extract obtained from Rhatany root by hydroalcoholic extractionwas applied onto the cornea of a bovine eye which had been previously incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured.

The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.

 

Hank’s Balanced Salt Solution (HBSS) was used as negative control. In all experiments the negative control showed no irritating effect on the cornea and the calculated mean IVIS (In Vitro Irritancy Score) was 1.23 (Exp. 1), 0.89 (Exp. 1b) and 1.24 (Exp. 1c) .

Undiluted dimethylformamide (DMF) was used as positive control. In all experiments the positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated mean IVIS was 73.43 (Exp. 1), 85.35 (Exp. 1b) and 91.85 (Exp. 1c).

 

In the first experiment under the conditions of the study, the test itemKrameria triandra extractshowed no effects on the cornea of the bovine eye. The calculated mean IVIS was 2.86 (Exp. 1).

In the second and third experiment under the conditions of this study, the test itemKrameria triandra extract obtained from Rhatany root by hydroalcoholic extraction showed effects on the cornea of the bovine eye. The calculated mean IVIS was 4.49 (Exp. 1b) and 3.02 (Exp. 1c).

The two out of three approach lead to the following assessment:

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage with the BCOP study only. In this case no prediction can be made. But as the test item itself is inhomogeneous, the result of the study has to be considered equivocal.

Categories Display