Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

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:
07 February - 14 February 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 guidelineopen allclose all
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:
Adopted July 26, 2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Commission Regulation (EU) No. 1152/2010 method B.47 (published in the Official Journal of the European Union L324, dated December 09, 2010)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Fatty acids, lanolin, lithium salts
EC Number:
268-919-1
EC Name:
Fatty acids, lanolin, lithium salts
Cas Number:
68154-72-3
IUPAC Name:
Fatty acids, lanolin, lithium salts
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: sponsor batch# SOAP 012-2
- Manufacture date of the lot/batch: Not reported
- Expiration date of the lot/batch: November 2022
- Purity test date: 13 December 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Physical characteristics: Beige to brown, solid
- Storage condition of test material: At ambient temperature (10 - 25°), container kept tightly closed and stored in a dry and well-ventilated place.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Vehicle: 0.9% sodium chloride solution
- Final dilution of a dissolved solid, stock liquid or gel: 10% (w/v)

FORM AS APPLIED IN THE TEST (if different from that of starting material): Suspended

OTHER SPECIFICS: The solid test item was suspended in a 0.9% sodium chloride solution with a final concentration of 10% test item (w/v) as recommended in the test guideline 437 for surfactant solids. 0.9% NaCl solution was used as the negative control and 1% NaOH solution in 0.9% NaCl solution as the positive control item.

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST MODEL
- Test model: Bovine eyes from cattle obtained from slaughterhouse at age 6-12 months
- Source: Hubert Bahlmann GmbH & Co. Versandschlachterei Speziamichfutterwerk KG 49699, Lindern, Germany
- Storage: Bovine tissues were stored in Hanks’ Balanced Salt Solution (HBSS) (GIBCO) containing penicillin at 100 IU/mL (GIBCO) and streptomycin at 100 µg/mL (GIBCO).
- Quality criteria: Eyes were examined for opacity, scratches and neovascularisation, only corneas from eyes free of defects were used. Prior to the assay, corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders, were also discarded.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 ± 1°C

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 the 10% test item suspension in 0.9% sodium chloride solution (w/v)
- Concentration (if solution): 10%

NEGATIVE CONTROL
- Negative control: Physiological saline solution (0.9% sodium chloride solution)
- Amount(s) applied (volume or weight): 750 µL

POSITIVE CONTROL
- Positive control: NaOH
- Amount(s) applied (volume or weight): 750 µL of the 1% NaOH solution in highly purified water
- Concentration (if solution): 1%
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
90 ± 5 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
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 (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL. 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.
The quality of each cornea was also evaluated at later steps in the assay. Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded. The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM), while preventing bubble formation. The corneal holder was equilibrated at 32±1°C for at least one hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer. A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment, solvent and positive control groups

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED
- Negative control: Physiological saline solution (0.9% sodium chloride solution)
- Amount(s) applied (volume or weight): 750 µL

POSITIVE CONTROL USED
- Positive control: NaOH
- Amount(s) applied (volume or weight): 750 µL of the 1% NaOH solution in highly purified water
- Concentration (if solution): 1%

APPLICATION DOSE AND EXPOSURE TIME
- Amount(s) applied (volume or weight with unit): 750 µL of the 10% test item suspension in 0.9% sodium chloride solution (w/v)
- Exposure time: 10 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the exposure period of 10 minutes (the recommended exposure time for surfactant solids) the test item, the negative and positive controls, were removed from each chamber. Subsequently, the epithelium was washed with EMEM containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.

- POST-EXPOSURE INCUBATION:
- Post-exposure preparation: To determine the corneal permeability 1 mL sodium fluorescein solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM.
- Incubation period: 90 ± 5 minutes
- Examination of corneas: Sodium fluorescein measurements (Tecan Sunrise Magellan Version 7.2)

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 resulting in opacity values measured on a continuous scale.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of (Tecan Sunrise Magellan Version 7.2 Microtiter plate reader) (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
After correcting the opacity and mean permeability (OD490) values for background opacity and the negative control permeability OD490 values, the mean opacity, and permeability OD490 values for each treatment group were combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The opacity and permeability values were also evaluated independently to determine whether the test item induced corrosivity or severe irritation through only one of the two endpoints.

DECISION CRITERIA:
A test item that induces an IVIS > 55 is defined as a corrosive or severe irritant.
The BCOP test method can also be used to identify chemicals that do not require classification for eye irritation or serious eye damage under the UN GHS classification system. When used for this purpose, the BCOP test method has an overall accuracy of 69% (135/169), a false positive rate of 69% (61/89), and a false negative rate of 0% (0/107), when compared to in vivo rabbit eye test method data classified according to the UN GHS classification system.
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) are >55 and ≤3, respectively.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
mean opacity (n=3), relative to negative control
Value:
-0.252
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Remarks:
(permeability)
Run / experiment:
Mean permeability score (n=3) relative to negative controls
Value:
0.002
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Calculated IVIS of 3 corneas exposed to strontium apatite copper-doped
Value:
-0.217
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Range of historical values if different from the ones specified in the test guideline: The most recent control data for GLP BCOP studies at LPT (2015-2017) are as follows:

Negative control (0.9% NaCl): presented as mean ± standard deviation (limits of acceptance)
IVIS: 0.579 ± 1.097 (-1.615 - 2.773)
Opacity: 0.062 ± 0.934 (-1.806 - 1.931)
Permeability: 0.043 ± 0.073 (-0.102 - 0.189)

Positive control (NaOH 1%): presented as mean ± standard deviation (limits of acceptance)
IVIS: 102.903 ± 21.283 (60.337 - 145.469)
Opacity: 72.668 ± 20.908 (30.852 - 114.484)
Permeability: 2.016 ± 0.415 (1.186 - 2.847)

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: TThe corneas treated with the negative control item 0.9% sodium chloride solution revealed a mean opacity value of <0.001 and a mean permeability value of 0.005 ± 0.003. The calculated IVIS value of <0.001 was well below the cut-off value of 3 (UN GHS no category).
- Acceptance criteria met for positive control: The corneas treated with the positive control item 1% NaOH in highly purified water revealed a mean opacity value of 68.858 ± 14.992 and a mean permeability value of 2.547 ± 0.266 compared to the solvent control. The calculated IVIS value of 107.068 ± 11.486 was within two standard deviations of the current historical mean and well above the cut-off value of 55.
- The acceptance criteria for the test were fulfilled.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the present test conditions Fatty acids, lanolin, lithium salts tested in the in vitro BCOP test method, had an IVIS value of <0.001, which is below the cut-off value of 3 (UN GHS no category) and consequently it is not classified as a severe irritant and is not corrosive according to UN GHS classification.
Executive summary:

Eye irritation is primarily defined by the extent of corneal injury; substances which damage the superficial epithelium may cause slight irritation, whereas further penetration to the corneal stroma or endothelium may induce mild or severe irritation, respectively. Eye irritation of the test item was evaluated in the Bovine Corneal Opacity and Permeability Test (BCOP, OECD 437), which utilises measurements of corneal opacity as an indicator of protein denaturation, swelling, vacuolation and tissue damage, and corneal fluorescein retention/leakage provides a measure of permeability in vitro.

A 10% suspension of the test item was applied to bovine corneas in 750 µL of physiological saline solution (0.9% NaCl) for 10 minutes, alongside positive and negative controls. Following treatment with Fatty acids, lanolin, lithium salts a mean opacity of <0.001 and a mean permeability value of 0.002±0.002 compared to the negative control were determined. The calculated IVIS of<0.001 is below the cut-off value of 3 (UN GHS no category) and consequently it is not classified as a severe irritant and is not corrosive according to UN GHS classification.

The BCOP (EU B.47; OECD 437) is an accepted in vitro test method to detect serious eye damage (Category 1 under CLP) and/or absence of effects requiring classification for serious eye damage/eye irritation (i.e. not classified under CLP), as described in the Annex to the EU Test Methods (TM) Regulation (Council Regulation (EC) No 440/2008). Conducted according to the aforementioned guidelines and GLP, the BCOP passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).