Registration Dossier

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:
weight of evidence
Study period:
2016-10-18 - 2016-11-15 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study
Remarks:
complementary eye irritation / corrosion study
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-03-06 - 2017-03-10 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Remarks:
complementary eye irritation / corrosion study
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:
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, (Adopted 28 July 2015).
Deviations:
no
GLP compliance:
yes
Species:
human
Strain:
other: not applicable
Remarks:
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm²) prepared from normal human keratinocytes (MatTek).
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability; Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
Test System
EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 23471 kit J)
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm²) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.
Rationale
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).
Source
MatTek Corporation, Ashland MA, U.S.A.
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): The solid test item (50.9 to 58.9 mg) was applied directly on top of the skin tissue; two tissues were treated with 50 µl Milli-Q water (negative control) and 2 tissues with 50 µl Methyl Acetate (positive control) respectively.
Duration of treatment / exposure:
6 hours ± 15 minutes
Duration of post- treatment incubation (in vitro):
18 hours ± 15 minutes
Number of animals or in vitro replicates:
2 / test group
Details on study design:
- Details of the test procedure used
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 64 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Application/Treatment of the Test Item: The test was performed on a total of 2 tissues per test item together with a negative control and positive control.
Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.
Two tissues were treated with 50 µl Milli-Q water (negative control) and 2 tissues with 50 µl Methyl Acetate (positive control) respectively.
At least 50 mg solid (with a glass weight boat) was added into the 6-well plates on top of the tissues. After the exposure period with the test item(6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. The test item remained on the skin tissue after washing. One of the skin tissues was detached half during rinsing. However, the tissue could be used during the further procedure. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 ml of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 ml of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.

- RhCE tissue construct used, including batch number
EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 23471 kit J)
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm²) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

- Doses of test chemical and control substances used
The solid test item (50.9 to 58.9 mg) was applied directly on top of the skin tissue. Two tissues were treated with 50 µl Milli-Q water (negative control) and 2 tissues with 50 µl Methyl Acetate (positive control) respectively.

- 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): spectrophotometer at 570 nm

- Description of the method used to quantify MTT formazan
Cell Viability Measurement
After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 ml MTT-medium (1.0 mg/ml). The tissues were incubated for 180 ± 10 minutes at 37°C.
After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labeled 6-well plate containing 2 ml isopropanol in each well so that no isopropanol is flowing into the insert. Formazan was extracted with 2 ml isopropanol for 2 hours at room temperature with gentle shaking.
The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.
The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post exposure incubation is less than or equal (≤) to 60%.

- Acceptability criteria:
The in vitro eye irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.
b) The mean relative tissue viability of the positive control should be <50% relative to the negative control.
c) The SD calculated from individual % tissue viabilities of the two identically treated replicates should be <20%.
d) The non-specific MTT reduction should be <= 50% relative to the negative control OD.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
Irritation parameter:
other: Mean Tissue Viability %
Run / experiment:
mean
Value:
31
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
potentially irritant or corrosive
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None stated

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
The positive control had a mean cell viability after 6 hours ± 15 minutes exposure of 34%. The absolute mean OD570 of the negative control tissues was within 0.8 and 2.5. The standard deviation value of the percentage viability of two tissues treated identically was less than 10%, indicating that the test system functioned properly.
Interpretation of results:
other: potentially irritant or corrosive in the EpiOcular™ test
Conclusions:
The study was conducted under GLP according to OECD guideline 492 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritating potential of the test substance to the eye in vitro.
The test was performed as a follow-up for an inconclusive BCOP study, see respective endpoint. As no conclusion on the definitive classification could be drawn from the ex vivo test, an additional in vitro test was performed.
In the present OECD 492 test, eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 31%. Since the mean relative tissue viability for (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate was below 60% it is considered to be potentially irritant or corrosive to the eye. Also here, no prediction on the classification can be made, as, according to the guideline, the EpiOcular EIT is not intended to differentiate between UN GHS Category 1 (serious eye damage) and UN GHS Category 2 (eye irritation). So generally, as within this in vitro test strategy no concrete conclusions on the classification can be drawn, further in vivo testing should be considered. However, as the registration is only intended to be in the tonnage band 1-10 tpa, only in vitro testing is mandatory under Annex VII, and no further in vivo testing to allow a definite classification is required. Hence, the result which can be drawn from the two available in vitro studies is inconclusive, no further testing according the REACH Regulation is required, and the substance does not need to be classified due to the lack of definitive evidence.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate. For this purpose (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate was topically applied on the Reconstructed Human EpiOcular™ Model.

The possible eye hazard potential of (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)benzenecarbothioate was tested through topical application for 6 hours.

The study procedures described in this report were based on the most recent OECD 492 guideline under GLP.

The test item (50.9 to 58.9 mg) was applied directly on top of the tissue for 6 hours ± 15 minutes. 

After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

The positive control had a mean cell viability of 34% after 6 hours ± 15 minutes exposure. The absolute mean OD570 of the negative control tissues was within 0.8 and 2.5. The standard deviation value of the percentage viability of two tissues treated identically was less than 10%, indicating that the test system functioned properly.

The test item did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). 

In addition to the normal procedure, two freeze-killed tissues treated with test item and one freeze-killed negative control treated tissue were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was 0.012% of the negative control tissues. The ODs of the test item treated viable tissues was corrected using the OD of the freeze-killed tissues.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 31%. Since the mean relative tissue viability for (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate was below or equal to 60% after 6 hours ± 15 minutes treatment it is considered to be potentially irritant or corrosive to the eye.

In conclusion, (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
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:
Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 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”(adopted July 26, 2013).
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Number of animals: nine eyes
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

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): 335 to 361 mg in experiment 1 and 305 to 389 mg in experiment 2

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µl (negative control)
Duration of treatment / exposure:
240 ± 10 minutes
Duration of post- treatment incubation (in vitro):
none
Number of animals or in vitro replicates:
3 replicates, 2 experiments
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES
3 replicates, 2 experiments

NEGATIVE CONTROL USED
physiological saline

POSITIVE CONTROL USED
20% (w/v) Imidazole solution

APPLICATION DOSE AND EXPOSURE TIME
The medium from the anterior compartment was removed and 750 µl of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (335 to 361 mg in experiment 1 and 305 to 389 mg in experiment 2). The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies)

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity=(I0/I - 0.9894) / 0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.
Permeability determinations: After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
In vitro irritancy score
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.
The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
In vitro score range UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. yes

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Remarks:
mean value
Run / experiment:
1st experiment
Value:
7.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Remarks:
mean value
Run / experiment:
1st experiment
Value:
0.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: permeability
Remarks:
mean value
Run / experiment:
1st experiment
Value:
0.485
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
mean value
Run / experiment:
2nd experiment
Value:
8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Remarks:
mean value
Run / experiment:
2nd experiment
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: permeability
Remarks:
mean value
Run / experiment:
2nd experiment
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
Test item induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.
Conclusions:
The study was conducted under GLP according to OECD guideline 437 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritating potential of the test substance to the eye in vitro.
Testing was conducted as an initial step in an in vitro top down testing strategy. (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate induced ocular irritation through the permeability endpoint only, resulting in a mean in vitro irritancy score of 7.6 and 8.0, in the first and second experiment, respectively, after 240 minutes of treatment. In the first experiment one cornea showed a negative response and two cornea showed irritation. These results were reproduced in the second experiment.
Since (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate induced an IVIS > 3 ≤ 55, no prediction on the classification can be made. However it should be noted that the in vitro irritancy score of 7.6 and 8.0 is rather close to the boundary value of 3, the IVIS score of one of the negative controls in the first experiment was also >3 (still within historical control data). It could be assumed that in in vivo testing the substance would rather reveal a negative outcome.
According to REACH Annex VII “Serious eye damage/eye irritation, in vitro: 8.2.1. If results from a first in vitro study do not allow a conclusive decision on the classification of a substance or on the absence of eye irritation potential, (an)other in vitro study/ies) for this endpoint shall be considered.” So, in order to allow a proper classification according to GHS, a second in vitro test for eye irritation / corrosion according to OECD 492 is conducted. For definitive conclusion, see the respective endpoint.
Executive summary:

Evaluation of the eye hazard potential of (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate was performed according to OECD 437 under GLP using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate was tested through topical application for approximately 240 minutes.

The study procedures described in this report were based on the most recent OECD guideline.

Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 125 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Since the test item induced IVIS scores spread over two categories (-1.7, 6.6 and 18.0 respectively), a second experiment was performed.

In the second experiment, the negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 149 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

(S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate induced ocular irritation through the permeability endpoint only, resulting in a mean in vitro irritancy score of 7.6 and 8.0, in the first and second experiment, respectively, after 240 minutes of treatment. In the first experiment one cornea showed a negative response and two cornea showed irritation. These results were reproduced in the second experiment.

Since (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.