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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
other: part of a testing Strategy
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
other: part of a testing strategy
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:
26 July 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: freshly slaughtered cattle; supplier: Schlachthof Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany
- Characteristics of donor animals: 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)
Remarks:
de-ionized
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL of de-ionized water

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 μL 100% ethanol / 100% dimethylformamide (positive controls, PC1 / PC2)
Duration of treatment / exposure:
4 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 test facility´s 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 of the 20% (w/v) test-substance preparation, 4 hours exposure time

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

- POST-EXPOSURE INCUBATION: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Yes.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader: Yes, OD490, 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 test facility 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:
5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
other: not identified as corrosive or severe irritant.
Conclusions:
Based on the results of the BCOP assay, the test substance was not identified as corrosive or severe irritant.
Executive summary:

The potential of the test substance to cause ocular irritation or 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 mean IVIS obtained in the BCOP test was 5.1, therefore the substance was not identified as corrosive or severe irritant.

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:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals / tissue source

Species:
human
Strain:
other: normal human derived epidermal keratinocytes
Details on test animals or tissues and environmental conditions:
Description of the cell system used: Reconstructed human cornea-like epithelium
- Model used: EpiOcular™ model (OCL-200)
- Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Certificate of authenticity: provided by the supplier
- Verification of tissue functionality and quality (performed by the supplier):
Tissue viability: acceptance criteria met
Barrier function: acceptance criteria met
Sterility: no contamination

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): ca. 50 μL of the undiluted test substance

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL, undiluted
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
2
Details on study design:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were immersed and swiveled three times in each of three beakers filled with sterile PBS.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
A direct interference was not observed.

NUMBER OF REPLICATE TISSUES PER TEST CHEMICAL AND CONTROLS: 2

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL MTT diluent
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm, without reference filter

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to the eye if the mean relative tissue viability with a test material is less than 55%.
- The test substance is considered to be non- irritant to the eye if the mean relative tissue viability with a test material is greater than 65%.
- In case of borderline results such as non-concordant replicate measurements and/or mean percent tissue viability of 55 - 65%, a second test should be considered as well as a third one in case of discordant results between the first two tests.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 492: 4The „borderline“-evaluation (60 ± 5%) was determined statistically using historic data of the test facility and hence considers the variance of the test method. This evaluation is an amendment to the evaluation provided in OECD Guideline 492.

ACCEPTANCE CRITERIA
- Negative control: Tissue viability is acceptable if the mean OD570 of the negative control (NC) is ≥ 0.8. The mean OD570 of the NC should not exceed 2.5.
- Positive control: Methyl acetate used as positive control (PC) usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.
- Variability: Two tissues were treated under the same conditions. A variability between the tissues is considered to be acceptable if the difference of the viability is ≤ 20%.

HISTORICAL DATA (see table 1)

Results and discussion

In vitro

Results
Irritation parameter:
other: mean viability [%]
Run / experiment:
Run 1, mean of 2 replicate tissues
Value:
51.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
other: irritant
Conclusions:
Using the currently available methods a single in vitro assay is not sufficient to cover the full range of eye irritating potential. In combination with the BCOP assay, which identified the test substance as not corrosive or severe irritant, the test substance is classified as GHS Cat. 2.
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 relative mean viability of the tissues treated with the test substance was 51.3%.

Based on the results of the BCOP and EpiOcular Tests and by applying the evaluation criteria described in section 3.10, it was concluded that 2-Methylbenzyl chloride shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.