Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion: Not irritating (OECD 439/GLP)

Serious eye damage/eye irritation: Not irritating (OECD 438/GLP)

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:
17 July 2017 - 15 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Willing New Material Technology Co.,Ltd.; 23161211201
- Expiration date of the lot/batch: 10 December 2017
- Purity: 97%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 ºC)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Adult donors
Justification for test system used:
The EPISKINTM (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
EPISKINTM (SM) (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-032, Expiry
Date: 14 August 2017) is a three-dimensional human epidermis model. The EPISKINTM (SM) plate contains up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.

EPISKINTM (SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKINTM (SM) test kits used in the present study) and are documented in Appendix 2.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (23.2-27.6°C).
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the 15 minutes incubation time, the EPISKINTM (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).

- Observable damage in the tissue due to washing: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution (prepared on 08 August 2017) was stored in refrigerator (2-8°C) protected from light. It was diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

After the 42 hours incubation, all EPISKINTM (SM) units (except the two living colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKINTM (SM) units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light, in a >95% humidified atmosphere.

After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate). The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.

The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 24072800, Serial Number: 0920-14, Date of calibration: 22 August 2016, calibration is valid until August 2018) at the required wavelength on each day before use.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Check-method for possible direct MTT reduction with test item
10 mg of test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in a shaking water bath for 3 hours protected from light, and then any colour change was recorded:
-Test items which do not react with MTT: yellow
-Test items reacting with MTT: blue or purple
After three hours incubation, yellow colour of the mixture was detected in the test tube. Thus, the test item did not react with MTT and therefore the use of additional controls was not necessary.

Check-method to detect the colouring potential of test-items

Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol* (simulating a tissue humid environment).
*Note: Water is the environment during exposure, isopropanol is the extracting solution.
Therefore, in addition to the normal procedure, two additional test item-treated living tissues were used for the non specific OD evaluation. This tissue followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.


PREDICTION MODEL / DECISION CRITERIA
The test item considered to be irritant to skin (Category 2), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control. If mean relative viability after 15 minutes exposure and 42 hours post incubation is greater than or equal (≥) to 50% of the negative control, the substance is not classified.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
As the test item was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 10 mg of the test item was applied evenly to the epidermal surface.

NEGATIVE CONTROL
50 μL of negative control (PBS) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).

POSITIVE CONTROL
50 μL positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
Duration of treatment / exposure:
15 minutes (± 0.5 min)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
TiBTD
Value:
93.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: SD (%)
Remarks:
4.6
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Positive Control
Value:
8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: SD (%)
Remarks:
2.3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Negative Control
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: OD (%)
Remarks:
3.8
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: As no colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.

- Colour interference with MTT: As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues were 0.008, Non Specific Colour % was calculated as 1.1% (see Table 1). This value was below 5%, therefore additional data calculation was not necessary.

ACCEPTANCE OF RESULTS:
After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.

The mean OD value of the three negative control tissues (0.751) was in the recommended range of 0.6 and 1.5. Standard deviation of the viability results for negative control samples was 3.8%.

The positive control treated tissues showed 8.0% viability, which is in the recommended range of 0 – 40%, demonstrating the proper performance of the assay.

The standard deviation of the viability results for positive control samples was 2.3%.

The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 4.6%.

The mean OD value of the blank samples (acidified isopropanol) was 0.047.

All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Historical control data are presented in Appendix 3.
Interpretation of results:
GHS criteria not met
Conclusions:
In this in vitro EPISKINTM (SM) model test with TiBTD, the results indicate that the test item is a non-irritant to skin.
Executive summary:

In an in vitro skin irritation assay in a human epidermal model EPISKINTM (SM) (17_053-043B), water-moistened reconstructed human epidermis tissue was exposed to 10 mg of TiBTD (97%) for 15 minutes (± 0.5 min). PBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and a 2 hour isopropyl alcohol extraction period followed. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

The colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing. The average viability of tissues treated by the test substance TiBTD was 93.1 % of negative control average value i.e. viability was > 50 %. The average viability of tissues treated by the positive control (5% SDS) was 2.3 % of negative control average value. According to these results, the test substance is not irritating.

This in vitro skin irritation study in the human epidermal model EPISKINTM (SM) is acceptable and satisfies the guideline requirement for an OECD 439 study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 July 2017 - 13 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Willing New Material Technology Co.,Ltd.; 23161211201
- Expiration date of the lot/batch: 10 December 2017
- Purity: 97%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 ºC)
Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)

Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.

After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within 2 hours of collection in each experiment.
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): 30 mg
Duration of treatment / exposure:
10 seconds
Number of animals or in vitro replicates:
3 animals
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluoresceintreated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve to short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.

The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during thenacclimatization and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes of this study. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
30 μLPhysiological saline (0.9% (w/v) NaCl)

POSITIVE CONTROL USED
30 mg Imidazole

APPLICATION DOSE AND EXPOSURE TIME
10 seconds

OBSERVATION PERIOD
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period:
The time of application was observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual of the test item if possible.

METHODS FOR MEASURED ENDPOINTS:
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit BP 900® slit-lamp microscope was used for the measurements.

- Macroscopic morphological damage to the surface:
Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the Investigator.

SCORING SYSTEM:
- Mean corneal swelling (%):Refer to Section 3.8.1 of the report
- Mean maximum opacity score: Refer to Section 3.8.2 of the report
- Mean fluorescein retention score at 30 minutes post-treatment: Refer to Section 3.8.3 of the report
Irritation parameter:
percent corneal swelling
Remarks:
75 mins
Run / experiment:
TiBTD Expt 1
Value:
1.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
percent corneal swelling
Remarks:
240 mins
Run / experiment:
TiBTD Expt 1
Value:
2.2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
cornea opacity score
Run / experiment:
TiBTD Expt 1
Value:
0.83
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class II
Irritation parameter:
fluorescein retention score
Run / experiment:
TiBTD Expt 1
Value:
0.33
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
percent corneal swelling
Remarks:
75 mins
Run / experiment:
TiBTD Expt 2
Value:
0
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
percent corneal swelling
Remarks:
240 mins
Run / experiment:
TiBTD Expt 2
Value:
1.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
cornea opacity score
Run / experiment:
TiBTD Expt 2
Value:
0.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
fluorescein retention score
Run / experiment:
TiBTD Expt 2
Value:
0.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
percent corneal swelling
Remarks:
75 mins
Run / experiment:
Positive control Expt 2
Value:
10.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class II
Irritation parameter:
percent corneal swelling
Remarks:
240 mins
Run / experiment:
Positive control Expt 2
Value:
28.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class III
Irritation parameter:
cornea opacity score
Run / experiment:
Positive control Expt 2
Value:
4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class IV
Irritation parameter:
fluorescein retention score
Run / experiment:
Positive control Expt 2
Value:
3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class IV
Irritation parameter:
other: All parameters
Run / experiment:
Negative control Expt 1 & 2
Value:
0
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Other effects / acceptance of results:
The results from Positive Control Experiement 1 were:
Mean maximum corneal swelling at up to 75 min - 8.0% ICE Class II
Mean maximum corneal swelling at up to 240 min - 24.1% ICE Class III
Mean maximum corneal opacity - 4.00 ICE Class IV
Mean fluorescein retention - 3.00 ICE Class IV

OTHER EFFECTS:
- Visible damage on test system:

Test item Expt 1: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 120 minutes after the post-treatment rinse.

Test item Expt 2: Test item was stuck on all cornea surfaces after the post-treatment rinse. The one cornea surface (1/3) was cleared at 30 minutes, and two cornea surfaces (2/3) were cleared at 75 minutes after the post-treatment rinse.

Positive control: Imidazole was stuck on all cornea surfaces after the posttreatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Positive control: Imidazole was stuck on all cornea surfaces after the posttreatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

ACCEPTANCE OF RESULTS:
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in each experiments. This study was considered to be valid.
Interpretation of results:
GHS criteria not met
Conclusions:
Based on these in vitro eye irritation assays in isolated chicken eyes with TiBTD, the test item was non-irritant, UN GHS Classification: No Category.
Executive summary:

In an in vitro eye irritation test in isolated chicken eyes (ICE) assay (17_053-038CS), isolated chicken eyes were exposed to TiBTD for 10 seconds in 2 independent experiments. Physiological saline (0.9% (w/v) NaCl) was used for the negative control and imidazole was used for the positive control. The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse.

The positive and negative controls gave the appropriate responses. In experiment I, no significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (severity 0.5 or 1) was observed on three eyes. No significant fluorescein retention change (severity 0.5) was noted on two eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. All cornea surfaces were cleared at 120 minutes after the post-treatment rinse. In experiment II, no significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on the test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on all three eyes. No significant fluorescein retention change (severity 0.5) was noted on all three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The one cornea surface was cleared at 30 minutes and two cornea surfaces were cleared at 75 minutes after the post-treatment rinse.

The ICE classes for TiBTD in experiment 1 were: 1xI (mean corneal swelling) 1xII (mean corneal opacity) 1xI (mean fluorescein retention).  TiBTD did not meet any of the criteria for Eye damage – Category 1 but did meet all the criteria for not classified. The ICE classes for TiBTD in experiment 2 were: 1xI (mean corneal swelling) 1xI (mean corneal opacity) 1xI (mean fluorescein retention). TiBTD did not meet any of the criteria for Eye damage – Category 1 but did meet all the criteria for not classified. TiBTD is not irritating to the eye based on the results from this experiment.

This in vitro eye irritation test in isolated chicken eyes (ICE) assay is acceptable and satisfies the guideline requirement for an OECD 438 study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion

The skin irritation/corrosion potential of TiBTD has been evaluated in 1 in vitro skin irritation test.

In an in vitro skin irritation assay in a human epidermal model EPISKINTM (SM) (OECD 439/GLP), water-moistened reconstructed human epidermis tissue was exposed to 10 mg of TiBTD (97%) for 15 minutes (± 0.5 min). PBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and a 2 hour isopropyl alcohol extraction period followed. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues. The colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing. The average viability of tissues treated by the test substance TiBTD was 93.1 % of negative control average value i.e. viability was > 50 %. The average viability of tissues treated by the positive control (5% SDS) was 2.3 % of negative control average value. According to these results, the test substance is not irritating.

Serious eye damage/eye irritation

The serious eye damage/irritation of TiBTD has been evaluated in 1 in vitro isolated chicken eyes (ICE) assay.

In an in vitro eye irritation test in isolated chicken eyes (ICE) assay (OECD 438/GLP), isolated chicken eyes were exposed to TiBTD for 10 seconds in 2 independent experiments. Physiological saline (0.9% (w/v) NaCl) was used for the negative control and imidazole was used for the positive control. The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse.

The positive and negative controls gave the appropriate responses. In experiment I, no significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (severity 0.5 or 1) was observed on three eyes. No significant fluorescein retention change (severity 0.5) was noted on two eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. All cornea surfaces were cleared at 120 minutes after the post-treatment rinse. In experiment II, no significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on the test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on all three eyes. No significant fluorescein retention change (severity 0.5) was noted on all three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The one cornea surface was cleared at 30 minutes and two cornea surfaces were cleared at 75 minutes after the post-treatment rinse.

The ICE classes for TiBTD in experiment 1 were: 1xI (mean corneal swelling) 1xII (mean corneal opacity) 1xI (mean fluorescein retention).  TiBTD did not meet any of the criteria for Eye damage – Category 1 but did meet all the criteria for not classified. The ICE classes for TiBTD in experiment 2 were: 1xI (mean corneal swelling) 1xI (mean corneal opacity) 1xI (mean fluorescein retention). TiBTD did not meet any of the criteria for Eye damage – Category 1 but did meet all the criteria for not classified. TiBTD is not irritating to the eye based on the results from this experiment.

The results from these tests are suitable to use in the human health hazard assessment.

Justification for classification or non-classification

Based on the available information in the dossier, the substance TiBTD (CAS No. 3064-73-1) does not need to be classified  for skin irritation/corrosion and does not need to be classified for serious eye damage/eye irritation when considering the criteria outlined in Annex I of 1272/2008/EC.