(S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate

Endpoint summary

Description of key information

Skin irritation/ corrosion: OECD 439, GLP compliance, tissue viability: 97%

Eye irritation 437: OECD 437, GLP compliance, IVIS > 3 ≤ 55

Eye irritation 492: OECD 492, GLP compliance, tissue viability: 31%

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-10-17 - 2016-11-28 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (adopted 28 July 2015)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 “In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test ". Official Journal of the European Union No. L142; Amended by EC No. 640/2012 OJ No. L193, 20 July 2012

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Irritant or corrosive: Yes
pH (1% in water, indicative range) 4.6 – 4.6 (determined by CRO)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France

Source strain:

other: n/a, human

Details on animal used as source of test system:

n/a

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2)
- Tissue batch number(s): Batch no.: 16-EKIN-047 (16-EKIN-042)
- Production date: TDS dated 2016-11-22 (2016-10-18), expiration date 2016-11-28 (2016-10-24)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 67 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.7 - 38.5°C). Temperature and humidity were continuously monitored throughout the experiment.
- Temperature of post-treatment incubation (if applicable): After incubation, killed epidermis was stored at ≤ -15°C.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The skin was moistened with 5 μl Milli-Q water. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. After incubation, cell culture inserts were dried carefully to remove excess medium.
- Observable damage in the tissue due to washing: none stated
- Modifications to validated SOP: none stated

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml in PBS
- Incubation time: 3h at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3 tissues per test item

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
not required, no interference

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): neat, 11.1 to 16.9 mg
- Concentration (if solution): The skin was moistened with 5 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl 5% SDS, the positive control was re-spread after 7 minutes contact time.

Duration of treatment / exposure:

After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item.

Duration of post-treatment incubation (if applicable):

After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

Number of replicates:

The test was performed on a total of 3 tissues per test item together with negative and positive controls.
For cell viability measurement, the amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate
Also, in cell viability measurement, in the initial assay, the untreated death skin had a viability of 82%. Therefore the assay was not considered valid and was repeated.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

relative mean tissue viability

Value:

97

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant. SD was 1%.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none stated
- Direct-MTT reduction: The non-specific reduction of MTT by the test item was -1.6% of the negative control tissues. Since the %NSMTT ≤ 0.0, there is no need to correct for this interaction.
- Colour interference with MTT: The test item was checked for colour interference in aqueous conditions and for possible direct MTT reduction by adding the test item to MTT medium. Because a colour change was observed by adding MTT-medium it was concluded that (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate did interact with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: OD570 values were within historical control data and the Standard Deviation value (SD) of the % viability was <=18.
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Interpretation of results:

GHS criteria not met

Conclusions:

The study was conducted under GLP according to OECD guideline 439 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 skin in vitro. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 97%. Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant. It is concluded that this test is valid and that (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations.

Executive summary:

In vitro skin irritation test was performed with (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)benzenecarbothioate using a human skin model according to OECD 439 under GLP.

This report describes the ability of (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM^TM)). The possible skin irritation potential of (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch Z900247673 of (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate was an off-white solid. Skin tissue was moistened with 5 µl of Milli-Q water and at least 10 mg of (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate was applied directly on top of the skin tissue for 15士0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The test item did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. The non­specific reduction of MTT by the test item was -1.6% of the negative control tissues. Since the %NSMTT < 0.0, there is no need to correct for this interaction.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15士0.5 minutes treatment with the test item compared to the negative control tissues was 97%. Since the mean relative tissue viability for the test item was above 50% after 15士0.5 minutes treatment the test item is considered to be non-irritant.

The positive control had a mean cell viability of 11% after 15士0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 17%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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 for cross-reference:

reference to other study

Remarks:

complementary eye irritation / corrosion study

Qualifier:

according to guideline

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. 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, in vivo testing is not foreseen under Annex VII. 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. As compromise the substance will be be classified as Cat. 2, Eye irritant according to Regulation (EC) No 1272/2008.

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.

Reference 2

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

Reason / purpose for cross-reference:

reference to other study

Remarks:

complementary eye irritation / corrosion study

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:

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

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.

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

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

In vitro skin irritation test was performed with test item using a human skin model according to OECD 439 under GLP. This report describes the ability of test item to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM^TM)). The possible skin irritation potential of test item was tested through topical application for 15 minutes. The study procedures described in this report were based on the most recent OECD and EC guidelines. Skin tissue was moistened with 5 µL of Milli-Q water and at least 10 mg of test item was applied directly on top of the skin tissue for 15士0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15士0.5 minutes treatment with the test item compared to the negative control tissues was 97%. Since the mean relative tissue viability for the test item was above 50% after 15士0.5 minutes treatment the test item is considered to be non-irritant.

 

Eye irritation

OECD 492

The objective of this study was to evaluate the eye hazard potential of test item. For this purpose test item was topically applied on the Reconstructed Human EpiOcular™ Model. 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. 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 test item was below or equal to 60% after 6 hours ± 15 minutes treatment it is considered to be potentially irritant or corrosive to the eye. EpiOcular EIT is not intended to differentiate between UN GHS Category 1 (serious eye damage) and UN GHS Category 2 (eye irritation). 

OECD 437

Evaluation of the eye hazard potential of test item was performed according to OECD 437 under GLP using the Bovine Corneal Opacity and Permeability test (BCOP test). The eye damage of test item was tested through topical application for approximately 240 minutes. The test item was used as delivered and added pure on top of the corneas. Test item 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 test item induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Justification for classification or non-classification

Based on data evaluation of skin irritation test, the test item was considered as non skin irritant. The relative mean tissue viability of three individual tissues after 15min of exposure to the test item and 42 hour post incubation is greater than 50% of the mean viability of the negative controls. Thus the test item does not need to be classified for skin irritation according to Regulation (EC) No 1272/2008.

For eye irritation, as within this in vitro test strategy no concrete conclusions on the classification can be drawn, further in vivo testing should be considered. However, in vivo testing is not foreseen under Annex VII. 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. Taking all information into account the substance will be be classified as Cat. 2 (H319), Eye irritant according to Regulation (EC) No 1272/2008 and therewith the possible hazard is sufficiently described.