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Toxicological information

Eye irritation

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Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017 - 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2017-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
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:
2013
Deviations:
yes
Remarks:
The prediction model used for evaluating the results was amended by "borderline" criteria indicating inconclusive test results. The "borderline" criteria are based on historic BASF data and consider the test facility specific variance of the test method.
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, D-55116 Mainz
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 5222A20170420
- Purity test date: 20 Apr 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: As the test substance was applied undiluted no preparation of the test substance in a vehicle was required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: undiluted liquid test substance was applied
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: freshly slaughtered cattle
- Characteristics of donor animals (e.g. age, sex, weight): age of the animals: minimum 12 months, maximum 60 months
- indication of any existing defects or lesions in ocular tissue samples: Corneas free of defects (opacity, scratches, pigmentation etc.)
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): 750 µL
- Concentration (if solution): undiluted liquid test substance, purity: 99.57%
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32°C for at least 1 hour. After the equilibration period, the medium in both chambers was replaced by fresh pre-warmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer.

QUALITY CHECK OF THE ISOLATED CORNEAS: Any corneas that showed macroscopic tissue damage or an opacity value < 550 opacity units were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups.(According to OECD TG 437, corneas that have an opacity value >7 or equivalent for the opacitometer and cornea holders used after an initial one-hour equilibration period are to be discarded. In the BASF opacitometer in combination with the used cornea holder set 2013-22 and 2013-24, the maximal initial opacity of >7 arises from I= 550 lux with Io= 641 lux).

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Yes

POSITIVE CONTROL USED: Yes

APPLICATION DOSE AND EXPOSURE TIME: Application of 750 µL undiluted test material (purity 99.57%), 10 min exposure time

POST-INCUBATION PERIOD: Yes, 2 hours.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3times.

- POST-EXPOSURE INCUBATION: Yes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Yes.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader]: Yes, OD490nm, SunriseTM Absorbance Reader.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: amendment to TG (refinement of decision criteria).
IVIS: < 1.5, Prediction: No classification for eye irritation.
IVIS: 1.5 – 4.5, Prediction: Borderline.
IVIS: > 4.5, < 45, Prediction: No prediction can be made for eye irritation, further testing with another suitable method is required.
IVIS: 45 - 65, Prediction: Borderline.
IVIS: > 65, Prediction: Ocular corrosive or severe irritant.
The “borderline“ evaluation (IVIS 3.0 ± 1.5 and 55.0 ± 10.0) was statistically determined by using historic BASF data and takes the test facility specific variance of the test method into account. This evaluation is an amendment to the evaluation provided in OECD Guideline 437
Irritation parameter:
in vitro irritation score
Run / experiment:
Run 1, mean of three single corneas
Value:
1.8
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency given.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: The mean opacity value and the mean IVIS of the positive control (ethanol = PC1) of the present study lies slightly out of the range of the data given above. However, as all other quality criteria of the test were met, this deviation is not considered to have any influence on the validity of the study.

Table 2: Results of the BCOP Test, in vitro irritancy score (IVIS) of the test substance, the NC and the PC.

Test substance identification Cornea-
No.
Opacity
per cornea
Permeability
per cornea
IVIS
per
cornea
per group
mean SD
test
substance
34 2.9 0.016 3.1 1.8 1.5
35 2.1 0.009 2.2
36 0.0 0.008 0.1
NC 16 15.8 0.001 15.8 8.4 6.5
17 4.5 0.003 4.6
18 4.6 0.006 4.7
PC1 19 13.9 0.641 23.5 22 1.4
20 13.6 0.552 21.9
21 13.0 0.515 20.7
PC2 22 103.3 0.377 108.9 105.9 4
23 98.0 0.626 107.4
24 89.8 0.774 101.4

NC: negative control

PC: positive control

Interpretation of results:
GHS criteria not met
Conclusions:
The test material was not identified as corrosive or severe irritant to the eye in the in vitro BCOP eye irritation test.
Executive summary:

The potential of the test substance to cause serious damage to the eyes was assessed by a single topical application of 750 µL undiluted test substance to the epithelial surface of isolated bovine corneas.

Three corneas were treated with the test substance for 10 minutes followed by a 2-hour post-incubation period.

In addition to the test substance, a negative control (NC; deionized water) and two positive controls (PC1 / PC2; 100% ethanol / 100% dimethylformamide) were applied to three corneas each.

Corneal opacity was quantitatively measured as the amount of light transmitted through the cornea. Permeability was quantitatively measured as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance.

The following results were obtained in the BCOP Test: Mean values for opacity, permeability and IVIS of the test substance (1.8%), the NC (8.4%), the PC1 (22%) and PC2 (105.9%).

Therefore, the test material was not identified as corrosive or severe irritant to the eye in the BCOP test.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, D-55116 Mainz

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 5222A20170420
- Purity test date: 20 Apr 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: As the test substance was applied undiluted no preparation of the test substance in a vehicle was required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: undiluted liquid test substance was applied

Test animals / tissue source

Species:
human
Strain:
other: Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia. Tissue model: OCL-200. Keratinocyte Strain: 4F1188.

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): 50 µL
- Concentration (if solution): undiluted, purity: 99.57%
Duration of treatment / exposure:
30 minutes
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used: EpiOcular Eye Irriation Test
- RhCE tissue construct used, including batch number: Tissue model OCL-200, Tissue Lot Number 23797
- Doses of test chemical and control substances used: test chemical (undiluted, purity 99.57%), sterile deionized water, neat methyl acetate
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Exposure (30 min, 37°C), post-soak immersion (12 min, 37°C), post-exposure incubation (2 hrs, 37°C)
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): two freeze-killed control tissues
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm, SunriseTM Absorbance Reader
- Description of the method used to quantify MTT formazan: Optical Density (OD) measurement
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: Mean tissue viability (% of negative control) < 55 = irritant, 55 - 65 = borderline, > 65 = non-irritant. The “borderline“ evaluation (60 ± 5%) was statistically determined by using historic BASF data and hence considers the variance of the test method. This evaluation is an amendment to the evaluation provided in OECD Guideline 492.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Yes (The mean viability value of the positive control of the present study lies slightly out of the range of historic control data. However, as all other quality criteria of the test were met, this deviation is not considered to have any influence on the validity of the study)
- Complete supporting information for the specific RhCE tissue construct used: Yes (Certificate of Analysis)
- Reference to historical data of the RhCE tissue construct: Yes (Certificate of Analysis)
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: proficiency given
- Positive and negative control means and acceptance ranges based on historical data: Yes
- Acceptable variability between tissue replicates for positive and negative controls: Yes
- Acceptable variability between tissue replicates for the test chemical: Yes

Results and discussion

In vitro

Results
Irritation parameter:
other: mean viability [%]
Run / experiment:
Run 1, mean of 2 replicate tissues
Value:
33.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
Mean viability value of positive control lies slightly out of the range of historic controls. However, as all other quality criteria of the test were met, this deviation is not considered to have any influence on the validity of the study.
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

Table 2: Results of the EpiOcular Test, individual and mean OD570 values, individual and mean viability values and inter-tissue variability

Test substance identification   tissue 1 tissue 2 mean Inter-tissue
variability [%]
NC mean OD570 1.547 1.420 1.483  
viability
[% of NC]
104.3 95.7 100 8.6
test
substance
mean OD570 0.539 0.454 0.497  
viability
[% of NC]
36.3 30.6 33.5 5.7
PC mean OD570 0.744 0.727 0.736  
viability
[% of NC]
50.1 49.0 49.6 1.1

NC: negative control

PC: positive control

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
The test material was identified as irritant to the eye in the in vitro EpiOcular eye irritation test.
Executive summary:

The potential of the test substance to cause ocular irritation was assessed by a single topical application of 50 µL undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™).

Two EpiOcular™ tissues were incubated with the test substance for 30 minutes followed by a 2-hour post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.

The following results were obtained in the EpiOcular™ eye irritation assay: The test substance is not able to directly reduce MTT. The mean viability of the tissues treated with the test substance was 33.5%.

Therefore, the test substance was identified as irritant to the eye in the EpiOcular test.